Studies were performed to further characterize male-specific hepatic recombinant micro-somal vitamin D-25-hydroxlase CYP2C11 expressed in baculovirus-infected insect cells and whether it also is a vitamin D-24-hydroxylase. 25- and 24-hydroxylase activities were compared to those of ten other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D₂, vitamin D₃, 1- hydroxyvitamin D₂ [1OHD₂] and 1-hydroxyvitamin D₃ [1OHD₃]. CYP2C11 had the greatest activity with these substrates except vitamin D₃ which had the same activity as 4 of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1OHD₃ >1OHD₂ >vitamin D₂ >vitamin D₃. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1OHD₂. CYP2C11 had the greatest activity. 24-Hydroxylation of 1OHD₃ was very low, and there was none with vitamin D₃. Only CYP2C11 24-hydroxylated vitamin D₂. Structures of vitamin D metabolites including 24-hydroxyvitamin D₂, 1,24(S)-dihydroxyvitamin D₂, and 1,24- dihydroxyvitamin D₃ were confirmed by HPLC and gas chromatography retention times, and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression and 24- and 25-hydroxylation of 1OHD₂. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared to uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is male-specific hepatic microsomal vitamin D-25-hydroxylase that hydroxylates vitamin D₂, vitamin D₃, 1OHD₂, and 1OHD₃. CYP2C11 also is a vitamin D 24-hydroxylase.