The formation of 1α, 25-dihydroxyvitamin D3 requires a 25-hydroxylation followed by a 1α-hydroxylation catalyzed by cytochrome P450 (CYP) enzymes in liver and kidney. The aim of this review is to give a brief summary of our research on the cytochrome P450 enzymes catalyzing the 25-hydroxylation and 1α-hydroxylation and to discuss the results in relation to other published literature on these enzymes. Two hepatic P450 enzymes catalyzing 25-hydroxylation of vitamin D3 exist in mammalian liver-one mitochondrial and one microsomal. The mitochondrial vitamin D3 25-hydroxylase is apparently identical with CYP27A, an obligatory enzyme in bile acid biosynthesis in liver. The microsomal 25-hydroxylase has been purified to apparent homogeneity from pig liver. The enzyme catalyzed 25-hydroxylation of vitamin D3, 1α-hydroxyvitamin D3, vitamin D2 and 1α-hydroxyvitamin D2. A cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase has been isolated in this laboratory. The primary structure of vitamin D3 25-hydroxylase shows 70-80% identity with members of the CYP2D subfamily and has been designated CYP2D25. Three different 1α-hydroxylating cytochromes P450 in kidney, ie CYP27A, CYP27B and a microsomal 1α-hydroxylase, have been described. Mitochondrial cytochrome P450, catalyzing 1α-hydroxylation and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3, was partially purified from pig kidney. Purification and inhibition experiments as well as experiments with a monoclonal antibody against CYP27A indicated that one single enzyme catalyzes both 1α-and 27-hydroxylation. Treatment of rats with a single iv dose of 1α, 25-dihydroxyvitamin D3 resulted in a marked suppression of CYP27A mRNA levels in kidney. The results suggest a role for CYP27A as a renal mitochondrial 1α-hydroxylase. Subsequently, several research groups reported the isolation of cDNA encoding mouse, rat and human kidney 25-hydroxyvitamin D3 1α-hydroxylase. The amino acid sequences deduced from these cDNA clones were similar but differed from that of CYP27A. This 1α-hydroxylase constitutes a new CYP27 subfamily, CYP27B. The expression of CYP27B was found to be influenced by vitamin D status and parathyroid hormone. Mutations in the CYP27B gene have been identified in patients with pseudovitamin D-deficiency rickets. A microsomal P450 catalyzing 1α-hydroxylation of 25-hydroxyvitamin D3 has been purified to apparent homogeneity from pig kidney. This finding demonstrate the presence of a microsomal 1α-hydroxylase in addition to the mitochondrial 1α-hydroxylases in kidney. The relative importance and regulation of the different renal 1α-hydroxylases in the bioactivation of vitamin D3 under normal and pathological conditions will be subject for future studies.