Streptomyces subtilisin inhibitors (SSIs) are produced by a wide variety of Streptomyces species. Streptomyces coelicolor A3(2) contains two genes, SCO0762 and SCO4010, encoding an SSI-like protein. Of these two genes, SCO0762 was transcribed actively throughout growth. Gene disruption of SCO0762 (mutant DeltaSCO0762) resulted in overproduction of extracellular protease activity, showing that SCO0762 serves as a modulator of extracellular protease activities. Mutant DeltaSCO0762 showed no apparent phenotypic changes in morphological differentiation, forming aerial hyphae and spores in the same time course as the parental strain. SCO4010 appeared to be a pseudogene, because mutant DeltaSCO4010 showed the same protease activity as the parental strain, probably due to amino acid replacement of one (Arg-60) of the important residues for SSI activity, and because the transcription of this gene was extremely low.