It's happened a few times--I think Spiderman has regretted killing (even if accidentally) a couple of villains. And a lot of his villains tend to be former friends, so... I've never been a big fan of Spiderman, so I can't recall. He obviously has an arch-nemesis and former friend in the Green Goblin, but he never killed him.

One that comes to mind is Robin (of Batman fame). He grew up to be a hero called Nightwing, and his arch-nemesis, Blockbuster (a former cop he knew--I don't know if I'd have called them friends, though), was killed by a vigilante he was working with. Nightwing had an chance to save Blockbuster but let him die, and he plunged into guilt afterward. Batman is a "no killing" type (though he's done it) so that added to Nightwing's guilt. He was a walking mess for a long time.

There was one time that Captain America had to kill but I can't find the reference to link you to it. He's a big "no killing" proponent, so it was a big deal.

Usually, though, comics keep the hero/villain friend dynamic in tact... example: Professor X and Magneto. They were close friends, and there was one time where Professor X shut down Magneto's mind. He didn't kill him, per se, but it was the closest I can recall that he ever came to killing Magneto.

/end rambling non-answer