As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products and determining the kinetics of D2 metabolism. Two new products were isolated by reverse-phase high-performance liquid chromatography: a dihydroxy metabolite with a hydroxyl group at C20 plus another unidentified position, and a trihydroxy metabolite identified by NMR as 17,20,24-trihydroxyvitamin D2. Kinetics of D2 metabolism was determined with substrate solubilized by 2-hydroxypropyl-beta-cyclodextrin or incorporated into phospholipid vesicles. In 2-hydroxypropyl-beta-cyclodextrin, D2 was hydroxylated at C20 with a k(cat)/K(m) 5-fold lower than that for cholesterol metabolism. 20-Hydroxyvitamin D2 was hydroxylated with a similar k(cat)/K(m) to D2, whereas 17,20-dihydroxyvitamin D2 was hydroxylated with a lower k(cat)/K(m) than that for D2 in 2-hydroxypropyl-beta-cyclodextrin. In vesicles, D2 displayed a high K(m) relative to that for cholesterol, but hydroxylation resulted in products that could be further hydroxylated with relatively low K(m) values. We conclude that P450scc catalyzes three sequential hydroxylations of D2 producing 20-hydroxyvitamin D2, 17,20-dihydroxyvitamin D2, and 17,20,24-trihydroxyvitamin D2, which dissociate from the active site of P450scc and accumulate in the reaction mixture. D2 metabolism occurs with lower efficiency (k(cat)/K(m)) than that observed for both cholesterol and D3 metabolism by P450scc.