It has been hypothesized that H-176 and E-177 are key residues in the catalytic activity of Pseudomonas aeruginosa alkaline protease. To test this possibility, we replaced H-175 with L-leucine and E-177 with L-glutamine using site-directed mutagenesis. The mutant enzyme proteins, produced by the Escherichia coli transformants harboring mutated genes, did not show any proteolytic activity without alteration of their molecular sizes compared to the wild-type enzyme protein.