Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the Km values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the Vmax values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes.