There has been some controversy over whether the 25-hydroxylation of vitamin D-3 is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D3 are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D-3, respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D-3 to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP21325 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP21325 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D-3. (C) 2003 Elsevier Science (USA). All rights reserved.