I most emphatically agree with everyone who votes Mark Hamill. That man just had a way with laughter...
As far as the debate between Nicholson and Ledger goes, though, it's little more difficult for me to decide. I'm all for comic accuracy, and if that's my only criteria, then I have to admit that Nicholson was far closer to the mark with his ability to be both vicious and hilarious at the same time, for the Joker is nothing if not a "comic genius". That somewhat goofy flair was definitely something Ledjoker was missing. And the end of the 1989 movie, however, I still couldn't quite get past the fact that was Jack Nicholson playing Jack Nicholson with face paint and a happy attitude. If comic accuracy is not my only criteria, if instead I look at which Joker fits best into the world he was filmed, then I have to say Ledger wins. Yes, most of his popularity comes from the fact he died before the movie came out (thus denying me, YET AGAIN, my dream of a Harley Quinn in a movie), but his creepyness just fits Nolan's dark, realistic world far better than Nicholson did in the weirdness of Tim Burton's mind.
So, seriously, Hamill wins. That is all.