How old is the new male kitten? Is he neutered?
You're doing the perfect thing in having him have his own room to begin with. I would just keep an eye out, and when they start paw-playing under the door or he gets VERY insistent on being let out, you might try opening the door and letting either new guy come out, or your females go in and check things out. If they growl or hiss, don't worry too much. They'll have to establish each other's place in the household. If they start fighting or hitting each other, yes, separate them again. Pretty soon, they'll get the picture that he's there to stay and they better learn to live with it, and they'll change their tune. Also be sure to give your females plenty of attention so they know they haven't been replaced or forgotten.
As far as length of time, it could be anywhere from a couple days to a couple weeks. It all depends on the kitties' personalities, and willingness to work with each other. It'll happen in their own time. You've already got a great start!