Okay, I'm possibly going to make a run for the most offensive entry here, but this is the line I always think of when it comes to off-hand hilarity. From South Park: Bigger, Longer & Uncut:

Kyle: Hey, Mole, be careful.

The Mole: Careful? Was my mother careful when she stabbed me in the heart with a clothes hanger while I was still in the womb?

I don't know if I can quite describe the amazing mixture of stunned silence and outrageous "Holy shit, I can NOT believe they went there!" that followed that line when I saw it in theatres. The long awkward pause after that line also really helps that joke sink in.

You can see it here at about 8:06
http://www.youtube.com/watch?v=lO2G6WgUq9c&feature=related