Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser(99) and His(212) as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp(210)/His(212) catalytic dyad. Activity is also strongly dependent on Asp(83) and Asp(85). Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.