If you mean they are in one location and you add ti Tunes, can you remove from this original location?
Yes.
When you add tio iTunes, files are copied to /Music/iTunes/ iTunes media/ folder.
You should not mess around inside this folder yourself. Let iTunes manage it.
You can move files to /Music/iTunes/iTunes media/Automatically add to iTunes/ folder and iTunes will move (not copy) the file to the correct location.
I keep a shortcut of this folder on my desktop adnsimply move files here and let iTunes take care of it.
Then I don't worry about have the original copy elsewhere on the drive.