His 67 residue is highly conserved among subtilisin family and changed to Arg in mammalian subtilisin-like proteins. To elucdate the structural and functional role of His 67 residue in subtilisin E, site-directed mutagenesis was carried out. Three mutants of subtilisin E with Ala 67, Glu 67 and Gly 67 were obtained. Each mutant was characterized in terms of proteolytic activity. Compared with wild type, proteolytic activity for Ala 67 mutant was about 30\%. Glu 67 and Gly 67 mutant did not show any detectable proteolytic activity on Nsuccinyl- L- Ala- L- Ala- L- Pro- L- Phe- p- nitroanilide but developed hallow zones onto the skim-milk plate. It is expected that His 67 residue in subtilisin E influences on catalytic efficiency.