When upgrading a site to the .Net Framework 4.0 I received a “Server Application Unavailable” after I asked one of our system administrators to change the site from the .Net Framework 2.0 CLR (2.0/3.5) to the .Net Framework 4.0 CLR. After some Google searching I came across a blog entry here that had some good tips (and had the tip that fixed my problem):
http://www.denalimultimedia.com/info/Articles.aspx?cid=20&topic=IIS-6-ASP-NET-4-0-page-not-found-404
In short for my problem, .Net 2.0 web apps and .Net 4.0 web apps cannot run in the same app pool. After creating an application pool for the new 4.0 site the problem was gone. The last thing that Frank mentions is that the Web Services Extension must be set to Allowed for the .Net Framework 4.0 (mine was by default).