An androst-4-ene-3,17-dione (AD)-producing mutant, AD1 and an androsta-1,4-diene-3,17-dione (ADD)-producing mutant, PD3 were derived from a cholesterol-, phytosterol-assimilating bacterium Mycobacterium sp. B-3805S and a cholesterol-assimilating bacterium Mycobacterium sp. NRRL B-3683, respectively, using nitrosoguanidine (NTG) mutagenesis. Production of AD or ADD from phytosterol by the two mutants was investigated in a 5-L surface-aeration microprocessor-controlled fermentor at 30 °C. It was found that the maximum conversion of phytosterol to AD or ADD was 67.9 or 70.6%, respectively. Control of glucose concentration in the fermentation culture is an important parameter that affects production of AD or ADD as a sole product from phytosterol. The major components of phytosterol such as campesterol, stigmasterol and β-sitosterol were proportionally consumed by the two mutants without nutrient preference. AD or ADD were isolated from the fermentation broth by the addition of Amberlite XAD-7 resin and were further purified by silica gel column chromatography. After crystallization in methanol, the purified AD or ADD was obtained as needle crystals, which were identical to authentic AD or ADD by physical and instrumental analyses.