We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vita-min D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Tri-hydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol.