Incubation of cholesterol with a bovine adrenocortical mitochondrial acetone-dried powder preparation yielded (22R)-22-hydroxycholesterol (I), (20R,22R)-20,22-dihydroxycholesterol(II), and pregnenolone (III) which were conclusively identified by combined gas chromatography-mass spectrometry. Incubations with [4-14C]cholesterol yielded I, II, and III with specific activities (determined from partial mass-spectral scans) not significantly different from those of the used substrate or the cholesterol reisolated after the incubation, demonstrating that the isolated compounds arose mostly, if not entirely, from the substrate cholesterol. Incubations in an 18O-enriched atmosphere yielded I, II, and III with 18O at position C-22, C-20 and C-22, and C-20, respectively, providing evidence that the hydroxyl groups of the side chain of I and II and the C-20 oxygen atom of III originated from molecular oxygen. The distribution of the oxygen atoms in II after incubation with 18O2 and 16O2 (devoid of 16O18O) proved that the hydroxyl groups of the side chain of II were introduced from two different molecules of oxygen, consistent with a sequential hydroxylation of cholesterol. No (20S)-20-hydroxycholesterol was found. Incubation of I in an 18O-enriched atmosphere afforded II and III with 18O at C-20.