Seven microbial peptide inhibitors-chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon-were tested for their efficiency to inhibit thermitase, a thermostable serine protease from Thermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with Ki values of 7×10-8M and 2×10-7M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN′ and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.