A steroid monooxygenase from cells of a fungus, Cylindrocarpon radicicola ATCC 11011, grown in the presence of progesterone has been purified by affinity chromatography on a pregnenolone-Sepharose column. The obtained enzyme was gel electrophoretically homogeneous and exhibited a molecular weight of about 115,000. SDS-gel electrophoresis revealed that the enzyme consisted of two equal-sized subunits with a molecular weight of 56,000. Sedimentation equilibrium analysis at 20 degrees C indicated that the enzyme protein behaved as a mixture of monomeric and dimeric subunit species. The enzyme contained one molecule of FAD in each subunit and exhibited absorption maxima at 375 and 440 nm. The monooxygenase catalyzed a Baeyer-Villiger type oxidation, i.e., oxygenative esterification of C21-20-ketosteroid to form an acetate ester of C19-17 beta-hydroxysteroid with consumptions of NADPH and molecular oxygen. The enzyme displayed a wide substrate specificity toward C21-20-ketosteroids, while it strictly required NADPH as the external electron donor in a ratio of 1:1:1 for ketosteroid:NADPH:molecular oxygen. Kinetic study showed the enzyme to have very high affinity for progesterone.