In a search for active-site residues of the Tsp protease, 20 positions were individually mutated to alanine, the mutant strains were assayed for growth defects in vivo, and the purified proteins were assayed for proteolytic activity in vitro. Alanine substitutions at three positions, Ser-430, Asp-441, and Lys-455, result in inactive proteases that have structures and substrate-binding properties similar to wild type, suggesting that the side chains at these positions participate in catalysis. Replacing Ser-430 with cysteine results in a partially active protease, which is inhibited by cysteine-modifying reagents. Replacing Asp-441 with asparagine does not significantly affect activity. However, other residues, including histidine and arginine, cannot functionally replace Lys-455. These data are consistent with a serine-lysine dyad mechanism, similar to those proposed for the LexA-like proteases, the type I signal peptidases, and the class A beta-lactamases.