We report an alternative, hydroxylating pathway for the metabolism of vitamin D2 in a cytochrome P450 side chain cleavage (P450scc; CYP11A1) reconstituted system. NMR analyses identified solely 20-hydroxyvitamin D2and 17,20-dihydroxyvitamin D2derivatives. 20-Hydroxyvitamin D2was produced at a rate of molÆmin )1 Æmol )1 P450scc, and 17,20-dihydroxy-vitamin D2 was produced at a rate of molÆmin )1 Æmol )1 .