The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases—the hepatic 25-hydroxylase, the renal 1α-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1α- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1α and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1α-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1α.