The physiological and nutritional factors regulating production of leupeptin, leupeptin-inactivating enzyme (LIE), and trypsin-like protease (TLP) in Streptomyces exfoliatus SMF13 were evaluated in batch and continuous cultures. Production of leupeptin in batch culture was closely associated with mycelial growth rate, but leupeptin was inactivated by LIE during the stationary phase. Production of LIE and TLP was induced when mycelial growth reached the stationary phase. Leupeptin production was reactivated by the external addition of glucose or leupeptin precursors (leucine or arginine) to cultures during the stationary phase, whereas the production of LIE and TLP was repressed. The production of leupeptin was related to mycelial growth rate, whereas the production of LIE and TLP was inversely related to mycelial growth rate in glucose-limited continuous culture. The production of leupeptin was enhanced under carbon-excess and phosphate-limited conditions, while the production of LIE and TLP was repressed. Production of LIE and TLP was strongly enhanced under the condition when both carbon and nitrogen were limited. Therefore, it is concluded that regulation of production of leupeptin is inversely related to that of LIE and TLP.