Did you add the AjaxControltoolkit dll to your Bin folder ?
One way of doing this is to create a new Ajax Web application. Open the web.config of that project and just compare the handlers that are in your new project to your existing web.config.
Another is to check out below :
Check out the Extensions in the web.config example below
You'll have to add the handlers.
<?xml version="1.0"?>
<!--
Note: As an alternative to hand editing this file you can use the
web admin tool to configure settings for your application. Use
the Website->Asp.Net Configuration option in Visual Studio.
A full list of settings and comments can be found in
machine.config.comments usually located in
\Windows\Microsoft.Net\Framework\v2.x\Config
-->
<configuration>
<configSections>
<sectionGroup name="system.web.extensions" type="System.Web.Configuration.SystemWebExtensionsSectionGroup, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35">
<sectionGroup name="scripting" type="System.Web.Configuration.ScriptingSectionGroup, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35">
<section name="scriptResourceHandler" type="System.Web.Configuration.ScriptingScriptResourceHandlerSection, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" requirePermission="false" allowDefinition="MachineToApplication"/>
<sectionGroup name="webServices" type="System.Web.Configuration.ScriptingWebServicesSectionGroup, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35">
<section name="jsonSerialization" type="System.Web.Configuration.ScriptingJsonSerializationSection, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" requirePermission="false" allowDefinition="Everywhere"/>
<section name="profileService" type="System.Web.Configuration.ScriptingProfileServiceSection, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" requirePermission="false" allowDefinition="MachineToApplication"/>
<section name="authenticationService" type="System.Web.Configuration.ScriptingAuthenticationServiceSection, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" requirePermission="false" allowDefinition="MachineToApplication"/>
<section name="roleService" type="System.Web.Configuration.ScriptingRoleServiceSection, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" requirePermission="false" allowDefinition="MachineToApplication"/>
</sectionGroup>
</sectionGroup>
</sectionGroup>
</configSections>
<appSettings/>
<connectionStrings>
<add name="efamroConnectionString" connectionString="Data Source=Sunil;Initial Catalog=efamro;User ID=sa;Password=Password1" providerName="System.Data.SqlClient"/>
<!--<add name="efamroConnectionString" connectionString="Data Source=130.117.82.172;Initial Catalog=efamro;User ID=flexi;Password=win4dup"
providerName="System.Data.SqlClient" />-->
</connectionStrings>
<system.web>
<!--
Set compilation debug="true" to insert debugging
symbols into the compiled page. Because this
affects performance, set this value to true only
during development.
-->
<compilation debug="true">
<assemblies>
<add assembly="System.Core, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
<add assembly="System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add assembly="System.Data.DataSetExtensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
<add assembly="System.Xml.Linq, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
<add assembly="System.Data.Linq, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
<add assembly="System.Web.Extensions.Design, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add assembly="System.Design, Version=2.0.0.0, Culture=neutral, PublicKeyToken=B03F5F7F11D50A3A"/>
<add assembly="System.Windows.Forms, Version=2.0.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/></assemblies>
</compilation>
<!--
The <authentication> section enables configuration
of the security authentication mode used by
ASP.NET to identify an incoming user.
-->
<authentication mode="Forms" />
<!--<forms loginUrl="~/CMS/Login.aspx">
<credentials passwordFormat="Clear">
<user name="admincms" password="adminCMS" />
</credentials>
</forms>
</authentication>
<authorization>
<deny users="?" />
</authorization>-->
<!--
The <customErrors> section enables configuration
of what to do if/when an unhandled error occurs
during the execution of a request. Specifically,
it enables developers to configure html error pages
to be displayed in place of a error stack trace.
<customErrors mode="RemoteOnly" defaultRedirect="GenericErrorPage.htm">
<error statusCode="403" redirect="NoAccess.htm" />
<error statusCode="404" redirect="FileNotFound.htm" />
</customErrors>
-->
<pages>
<controls>
<add tagPrefix="asp" namespace="System.Web.UI" assembly="System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add tagPrefix="asp" namespace="System.Web.UI.WebControls" assembly="System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
</controls>
</pages>
<httpHandlers>
<remove verb="*" path="*.asmx"/>
<add verb="*" path="*.asmx" validate="false" type="System.Web.Script.Services.ScriptHandlerFactory, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add verb="*" path="*_AppService.axd" validate="false" type="System.Web.Script.Services.ScriptHandlerFactory, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add verb="GET,HEAD" path="ScriptResource.axd" type="System.Web.Handlers.ScriptResourceHandler, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" validate="false"/>
</httpHandlers>
<httpModules>
<add name="ScriptModule" type="System.Web.Handlers.ScriptModule, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
</httpModules>
<siteMap defaultProvider="SqlSiteMapProvider">
<providers>
<add name="SqlSiteMapProvider" type="SqlSiteMapProvider"/>
</providers>
</siteMap>
</system.web>
<system.codedom>
<compilers>
<compiler language="c#;cs;csharp" extension=".cs" warningLevel="4" type="Microsoft.CSharp.CSharpCodeProvider, System, Version=2.0.0.0, Culture=neutral, PublicKeyToken=b77a5c561934e089">
<providerOption name="CompilerVersion" value="v3.5"/>
<providerOption name="WarnAsError" value="false"/>
</compiler>
<compiler language="vb;vbs;visualbasic;vbscript" extension=".vb" warningLevel="4" type="Microsoft.VisualBasic.VBCodeProvider, System, Version=2.0.0.0, Culture=neutral, PublicKeyToken=b77a5c561934e089">
<providerOption name="CompilerVersion" value="v3.5"/>
<providerOption name="OptionInfer" value="true"/>
<providerOption name="WarnAsError" value="false"/>
</compiler>
</compilers>
</system.codedom>
<!--
The system.webServer section is required for running ASP.NET AJAX under Internet
Information Services 7.0. It is not necessary for previous version of IIS.
-->
<system.webServer>
<validation validateIntegratedModeConfiguration="false"/>
<modules>
<remove name="ScriptModule"/>
<add name="ScriptModule" preCondition="managedHandler" type="System.Web.Handlers.ScriptModule, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
</modules>
<handlers>
<remove name="WebServiceHandlerFactory-Integrated"/>
<remove name="ScriptHandlerFactory"/>
<remove name="ScriptHandlerFactoryAppServices"/>
<remove name="ScriptResource"/>
<add name="ScriptHandlerFactory" verb="*" path="*.asmx" preCondition="integratedMode" type="System.Web.Script.Services.ScriptHandlerFactory, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add name="ScriptHandlerFactoryAppServices" verb="*" path="*_AppService.axd" preCondition="integratedMode" type="System.Web.Script.Services.ScriptHandlerFactory, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
<add name="ScriptResource" preCondition="integratedMode" verb="GET,HEAD" path="ScriptResource.axd" type="System.Web.Handlers.ScriptResourceHandler, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
</handlers>
</system.webServer>
<runtime>
<assemblyBinding xmlns="urn:schemas-microsoft-com:asm.v1">
<dependentAssembly>
<assemblyIdentity name="System.Web.Extensions" publicKeyToken="31bf3856ad364e35"/>
<bindingRedirect oldVersion="1.0.0.0-1.1.0.0" newVersion="3.5.0.0"/>
</dependentAssembly>
<dependentAssembly>
<assemblyIdentity name="System.Web.Extensions.Design" publicKeyToken="31bf3856ad364e35"/>
<bindingRedirect oldVersion="1.0.0.0-1.1.0.0" newVersion="3.5.0.0"/>
</dependentAssembly>
</assemblyBinding>
</runtime>
</configuration>