Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6β-hydroperoxycholest-4-en-3-one and 5α-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6β-hydroperoxycholest-4-en-3-one.