Go to your computer's System Tray and try ejecting. If you can't eject, there is an application that is trying to access your iPod.
Go to http://technet.microsoft.com/en-us/sysinternals/bb896653.aspx and download ProcessExplorer.
Go to http://support.microsoft.com/kb/555665 and follow the instructions under Resolution.
Note that this is a good, general process any time you can't access any file.
In my case an update to RealPlayer installed a program called RecordingManager that was accessing my iPod.