On my local machine, I work on multiple web sites and run them under IIS under a \"Default\" web site. That way I can access the sites through this type of URL: http://localhos
I've encountered this error in one of two scenarios, both of which were due to the applications being sub-directories of the overall "Default" application:
Sub-Directories also need to be a working Application
You've already noted that the directory is flagged as an application, however for completeness you can double check that the application is also working, is able to execute scripts and has permission to the files in it's directories.
Nested web.config files
This is the problem that I think you're actually experiencing:
Ensure that your hierarchy of web.config files, including any in the "Default" application above your development applications, are not interfering with each other. Remember that as a sub-directory of "Default" your application may still be affected by settings in the "Default" application above it.