Hopefully this will help someone else since I've seen it happen a few times.
Using ArcGIS Server 10.0 and Desktop 10.0. Using a mapServer type service on ArcGIS Online, features cannot be identified on a certain layer within the service. We know that ArcGIS Online sends a URL to our local server as part of the identify operation. Going into Chrome's developer tools, then copying and pasting the identify operation URL into a new browser session results in an error 400, with no helpful details.
The mapServer service comes from our local instance of ArcGIS Server. As a test, I added the mapServer layer to ArcMap and tried to identify a feature. Identify operations work in ArcMap. But not in ArcGIS Online - strange.
In an attempt to fix this, i restarted the service, removed and re-added the layer in the ArcGIS Online map. Still, cannot identify features. Same error 400 is returned.
Using the same layers, I rebuilt the MXD, created a new service, then added it to the ArcGIS Online map. Now identify operations are working just fine. Since the rebuilt MXD solved the issue, I am guessing there had to be corruption in the MXD.
This same series of events has played out several times. At least we know how to correct the problem.