The activation of vitamin D requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation. Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase. Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D-3 25-hydroxylase. A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D-2. CYP2R1 hydroxylated vitamin D-2 at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27. The K-m and k(cat) values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D-3 were 0.45 muM and 0.97 min(-1), respectively. The k(cat)/K-m value of CYP2R1 was 26-fold higher than that of CYP27A1. These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans. (C) 2004 Elsevier Inc. All rights reserved.