Scott Rendell put non-league Cambridge into the FA Cup third round as his 26th-minute penalty beat Blue Square Premier rivals Weymouth.
Rendell converted the spot-kick for his 15th goal of the season after he had been fouled by Justyn Roberts.
Weymouth keeper Jason Matthews had made a superb early save from Gavin Hoyte but Stephen Reed later cleared from Paolo Vernazza to preserve the lead.
Anton Robinson's late corner hit the bar for Weymouth but Cambridge held on.
Cambridge Utd: Potter, Morrison, Peters, Hoyte, Gleeson, Wolleaston (Convery 86), Carden, Reed, Pitt (Knights 64), Rendell, Boylan (Fortune-West 72).
Subs Not Used: Coulson, Willmott.
Goals: Rendell 26 pen.
Weymouth: Matthews, Doe, Young, Dutton, Hart, Barti, Browning (Crittenden 63), Robinson, Vernazza (Douglas 78), McCallum (Carmichael 85), Beavon.
Subs Not Used: Stewart, Weatherstone.
Booked: Browning, Douglas.
Ref: P Gibbs (West Midlands).