Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1alpha-hydroxylase contains the component CYP27B1, which completes the activation pathway to the hormonal form 1alpha,25-dihydroxyvitamin D(3). A five-step inactivation pathway from 1alpha,25-(OH)(2)D(3) to calcitroic acid is attributed to a single multifunctional CYP, CYP24A1, which is transcriptionally induced in vitamin D target cells by the action of 1alpha,25-(OH)(2)D(3). On the basis of alignments and crystal structures of other CYPs, homology models of vitamin-D-related CYPs have been generated. Two human forms of rickets caused by mutations of CYP2R1 and CYP27B1, as well as mouse knockout models of CYP27A1, CYP27B1 and CYP24A1, are helping us to establish the full in vivo physiological roles of the vitamin-D-related hydroxylases.