That bit in the middle of The Fellowship of the Ring where the cave troll pops out from behind the rock pillar gets me every time. Thing is, I knew it was coming even the first time I saw the movie–they totally telegraph it, but somehow I think that only heightens the effect. I read once about an experiment where it was demonstrated that people are more startled by a sudden loud sound if a soft sound precedes it, so there may be something to this.
The facehugger in Alien.
Bilbo Baggins going for the ring at Rivendel. I think some of the people in the audience browned their trousers at that moment in the film.
The very best BOO moment ever was near the end of Wait Until Dark. It was so good, I can’t remember a single other thing about the movie.
The ending of Carrie. Holy shit.
Twilight Zone: The Movie. “You wanna see something really scary?”
I came into this thread to contribute the above BOO moment.
I do remember lots of stuff from this fine oldie, and still grab the arms of my chair when
Alan Arkin leaps out and grabs Audrey Hepburn by the ankle.
In “The House on Haunted Hill” (the good one with Vincent Price, not the crappy re-make), when Nora is in the basement. She’s bent over listening, and then she straightens up, and EEEEK! There’s a crone behind her!
When the body dressed as Batman hits the Mayor’s window in “The Dark Knight”. Extra-startling since it’s not something you expect in a non-horror movie. I didn’t anyway.
The first time you see a ghost walk by in Sixth Sense is a good one.
The underwater scene in Jaws, in which the corpse’s head pops out of the bite hole in the boat.
I figured USCDiver had already covered this one when I opened the thread.
The cat in Alien. 
In the American remake of ‘The Ring’ where it flashes to the image of the dead girl in the closet
From what I remember, I think that scene made me literally wet my pants when I was nine. I watched again a couple of years ago (the good one), and I probably shouldn’t have. I thought it was lame, lame, lame.
The truck-driver lady in Pee-Wee’s Big Adventure.
In Signs, when the alien walks by on the home video footage that’s on the news.
In Disturbia, when they’re watching the film from the killer’s house and see the dead face in the vent.
Mulholland Drive - when the bum behind Winkies appears and scares Dan to death.
The calligraphy lesson in The Eye
Had to pause the DVD for a trouser change.
I second this one and the Carrie ending. Even after the first time they stay scary.
Near the begining of the movie The Thing, with the Dog. Also the CPR scene much later.
The twilight zone episode on the airplane with William Shatner, the window. Redone decades later by john lithgow. In third rock from the sun, they both make a a very funny reference to that airplane scenario.