What a great thread! I have a million of 'em... kind of a hobby of mine.
"Where's the fetus gonna gestate?! You gonna keep it in a box?!" from Life of Brian
"Deserves got nothing to do with it." from Unforgiven just before the bad guy gets it.
"See what happens when you pay for stuff man?" from After Hours when Cheech thinks his new statue has been stolen.
"Who am I?! There's a sign outside that says 'Lou's Tavern'. I'm f***in' Lou... Who the f*** are you?" is the audiences introduction to Lou in Fight Club
"Fra-jee-lay! It must be Italian..." from A Cristmas Story when dad gets his major award.
...but my all time favorite is a tossup between two from the same scene in It's a Wonderful Life;
"Look... We serve hard drinks here for men who wanna get drunk fast. We don't need no characters hangin' round to give the place atmosphere." Nick says to George and Clarence just before he kicks them out with the classic "That's it! Out you two pixies go, troo the door or troo the winda!"
Actually, my favorite line isn't really a line. It's the look on the delivery man's face when the father asks a second time what was in the box being delivered in A Christmas Story. The look on his face is to me the best delivery of a "line" in movie history.