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To examine cholesterol degradation by Bacillus subtilis SFF34.Cholesterol degradation and cholesterol oxidase production by B. subtilis SFF34 were investigated in a medium containing 0·2% cholesterol. In addition, the oxidized product of cholesterol by the purified cholesterol oxidase was detected using a gas chromatograph. Cholesterol oxidase production reached its maximal level (3·14 U ml−1) after 24 h of incubation in the cholesterol medium. The residual cholesterol content reduced to 0·98 mg g−1 after 60 h of cultivation in the cholesterol medium. Two cholesterol oxidases were purified from the culture supernatant fluid and their reaction product against cholesterol was identified as 4-cholesten-3-one.B. subtilis SFF34 degraded cholesterol and produced a high level of extracellular cholesterol oxidase.Bacillus subtilis will be very useful for the reduction of cholesterol in many fermented foods and as a source of cholesterol oxidase.