The most difficult boss I've ever played against would be the SA-X from Metroid Fusion. However, I'm not talking about when you battle her right before the end of the game...I'm talking about when you have to run get past her in Sector 2, after you turn on the auxilary power...sheesh, she's annoying (and I have fairly lousy aim with the missles, which could explain a lot...). If you want to know who I feel was the most difficult boss that I actually battled and not just ran from, I would probably say Dark Link from Zelda 2.