Genetic polymorphisms in VKORC1 and CYP2C9, genes controlling vitamin K(1) (VK1) epoxide reduction and (S)-warfarin metabolism, respectively, are major contributors to interindividual variability in warfarin dose. The V433M polymorphism (rs2108622) in CYP4F2 has also been associated with warfarin dose and speculatively linked to altered VK1 metabolism. Therefore, the purpose of the present study was to determine the role of CYP4F2 and the V433M polymorphism in the metabolism of VK1 by human liver. In vitro metabolic experiments with accompanying liquid chromatography-tandem mass spectrometry analysis demonstrated that recombinant CYP4F2 (Supersomes) and human liver microsomes supplemented with NADPH converted VK1 to a single product. A screen of all commercially available P450 Supersomes showed that only CYP4F2 was capable of metabolizing VK1 to this product. Steady-state kinetic analysis with recombinant CYP4F2 and with human liver microsomes revealed a substrate K(m) of 8 to 10 microM. Moreover, anti-CYP4F2 IgG, as well as several CYP4F2-selective chemical inhibitors, substantially attenuated the microsomal reaction. Finally, human liver microsomes genotyped for rs2108622 demonstrated reduced vitamin K(1) oxidation and lower CYP4F2 protein concentrations in carriers of the 433M minor allele. These data demonstrate that CYP4F2 is a vitamin K(1) oxidase and that carriers of the CYP4F2 V433M allele have a reduced capacity to metabolize VK1, secondary to an rs2108622-dependent decrease in steady-state hepatic concentrations of the enzyme. Therefore, patients with the rs2108622 polymorphism are likely to have elevated hepatic levels of VK1, necessitating a higher warfarin dose to elicit the same anticoagulant response.