Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents, This cholesterol oxidase produces 6 beta -hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O-2 and the formation of one mole of H2O2. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.