Let me make some guesses: is this is 9.0.1 or above, on IIS 7 (not running in IIS 6 compatibility mode), and had you chosen to add “all sites” when installing CF or later running the web server config tool? If so, it used to be that in doing that, any newly added site would inherit CF settings, but with the above configuration you can no longer rely on that. Instead, you have to rerun the web server config tool to again add “all sites”.
This is indeed a radical change in behavior from prior release. It is in fact discussed in the installer (if you do “all sites” during installation), as well as in the CF Admin and Config manual, and in the online help for the CF admin. But there’s not too much talk about it, so people only trip over it when they move to IIS 7 (and 9.0.1 or above, which was the first release to formally support IIS 7 other than in IIS 6 compat mode.)
Let us know if that helps, or if, perhaps this is NOT the problem, in which case it could be any number of things. Even then, if you run the web server config tool and connect that site to CF (or again redo the “all sites” config), does it help?