The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.