1. Where is the file that is being requested to stored on the file system (e.g. c:\inetpub\....)? I'm just asking to make sure that the virtual directory is actually pointing to the correct path.

 

2. Open IIS go to the website in question, right click and select properties. If the Application Name is blank then click the create button next to it. If it isn't blank then one has already been created for it.