We report an alternative, hydroxylating pathway for the metabolism of vitamin D-2 in a cytochrome P450 side chain cleavage (P450scc; CYP11A1) reconstituted system. NMR analyses identified solely 20-hydroxyvitamin D-2 and 17,20-dihydroxyvitamin D-2 derivatives. 20-Hydroxyvitamin D-2 was produced at a rate of 0.34 mol.min(-1).mol(-1) P450scc, and 17,20-dihydroxyvitamin D-2 was produced at a rate of 0.13 mol.min(-1).mol(-1). In adrenal mitochondria, vitamin D-2 was metabolized to six monohydroxy products. Nevertheless, aminoglutethimide (a P450scc inhibitor) inhibited this adrenal metabolite formation. Initial testing of metabolites for biological activity showed that, similar to vitamin D-2, 20-hydroxyvitamin D-2 and 17,20-dihydroxyvitamin D-2 inhibited DNA synthesis in human epidermal HaCaT keratinocytes, although to a greater degree. 17,20-Dihydroxyvitamin D-2 stimulated transcriptional activity of the involucrin promoter, again to a significantly greater extent than vitamin D-2, while the effect of 20-hydroxyvitamin D-2 was statistically insignificant. Thus, P450scc can metabolize vitamin D-2 to generate novel products, with intrinsic biological activity (at least in keratinocytes).