Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1α-hydroxyvitamin D₂ (1αOHD₂) as substrate. The ratio of rates of 24-hydroxylation of 1α-hydroxyvitamin D₃ (1αOHD₃), 1αOHD₂, and vitamin D₂ by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D₂, 1,24(S)-dihydroxyvitamin D₂, and 1,24-dihydroxyvitamin D₃ were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1αOHD₂ by CYP3A4 correlated significantly with 6β-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, α-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1αOHD₂ and 1αOHD₃ were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D₂, 1αOHD₂, and 1αOHD₃.