Me he encontrado con este problema al actualizar una aplicación web de .NET 2.0 a 3.5.
Compruebe que su web.config esté configurado correctamente para .NET 3.5. Añadí/modifican los siguientes:
<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>
<assemblies>
<!--<add assembly="System.Web.Extensions, Version=1.0.61025.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>-->
<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.Xml.Linq, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
<add assembly="System.Data.DataSetExtensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=B77A5C561934E089"/>
</assemblies>
<httpHandlers>
<remove verb="*" path="*.asmx"/>
<add verb="*" path="*.asmx" type="System.Web.Script.Services.ScriptHandlerFactory, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35" validate="false"/>
<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>
<pages enableSessionState="true" validateRequest="true">
<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>
<httpModules>
<add name="ScriptModule" type="System.Web.Handlers.ScriptModule, System.Web.Extensions, Version=3.5.0.0, Culture=neutral, PublicKeyToken=31BF3856AD364E35"/>
</httpModules>
Parece ser un duplicado. Refiérase a la respuesta de Mitchell aquí: http://stackoverflow.com/questions/582492/working-with-vs2008-3-5-asp-ajax-site-on-a-2-0sp1-ajax-extesnion-1-0-server –