Yes, you can. If you installed .NET 3.5. Otherwise, you need to update your site. It also can be depended on the configuration setting in your web.config. And if you use IIS 7.0 and running the 3.5 application in 4.0 framework. You need to change and put your application in different application pools.
Here's a link metioned about it:(in paragraph "To manually upgrade an application's Web.config file from the .NET Framework 3.5 to the .NET Framework 4")http://msdn.microsoft.com/en-us/library/dd483478.aspx
And check ASP.NET Side-by-Side Execution Overview, maybe you will have a clear picture.http://msdn.microsoft.com/en-us/library/a99txfy5.aspx
Hope this can help you.