Readyboost file refuses to leave my USB

[Terry Tortoise]

I have a copy of this on the USB I use for backing up certain files. It is V. Large and I want the space back but it won't let me delete it (it's only a file) saying 'It is in use'. I can't find it in use anywhere. Ideas for its murder?

 

[TrainableMan]

Try Unlocker.