Cytochrome P-45Oscc as isolated is a cholesterol-depleted low-spin haemoprotein; addition of cholesterol results in formation of a high-spin complex. Cytochrome P-45Oscc -cholesterol is a one-electron acceptor on titration with NADPH. Cytochrome P-45Oscc -cholesterol can be anaerobically reduced to the ferrous state which, on oxygenation, forms an oxygenated cytochrome P-45Oscc -cholesterol complex. This oxygenated complex in the absence of adrenodoxin autoxidises to ferric cytochrome P-45Oscc -cholesterol without oxidation of cholesterol. The decay of the oxygenated complex is first-order, k = 9.3 x s-' at 4 "C. The rate of autoxidation is influenced by pH, ionic strength and the chemical nature of bound sterol. The activation energy of autoxidation is 75 kJ mol-'. Addition of equimolar amounts of adrenodoxin to cytochrome P-45Oscc-cholesterol followed by stoichiometric reduction under anaerobic conditions and subsequent oxygenation, allows single catalytic turnover cycles of cytochrome P-45Oscc to be observed. This has led to detection of intermediates in the conversion of cholesterol to pregnenolone and a precursor/product sequence of cholesterol -+ 22-hydroxycholesterol -20,22-dihydroxycholesterol -+ pregnenolone has been established. Addition of oxidised adrenodoxin to oxygenated cytochrome P-45Oscc -cholesterol results in formation of 22-hydroxycholesterol.Cytochrome P-450 acts as the terminal oxidase of the electron-transport chain associated with the side-chain cleavage reaction of cholesterol to pregnenolone in adrenal cortex mitochondria [l]. In these mitochondria, the cytochrome P-450 (cytochrome P-45Oscc) associated with the side-chain cleavage reaction of cholesterol appears to be distinct from the cytochrome P-450 (cytochrome P-450,,,) associated with 1 1P-hydroxylation of deoxycorticosterone and these cytochromes have been partially resolved [2 -41.Cytochrome P-45OCA, is the terminal oxidase of the electron transport chain associated with hydroxylation of camphor to exo-5-hydroxy-camphor in Pseudomonas putida [5]. This cytochrome has been purified and crystallised [6] and a reaction cycle postulated [7, 81 for the catalytic turnover of the cytochrome to form a monohydroxylated product. However, the conversion of cholesterol to pregnenolone, catalysed by cytochrome P-45Oscc, appears to be a complex series of reactions which may involve stable intermediates. Trivial Names. Cholesterol, S-cholesten-'3B-ol; pregnenolone, 3B-hydroxy-5-pregnen-20-one; deoxycorticosterone, 21-hydroxy-4-pregnene-3