Nicotine is the major addictive agent in tobacco; it is primarily metabolized by 5′-oxidation. 4-(Methylnitrosamine)-1-(3-pyridyl)-1- butanone (NNK), a potent lung carcinogen generated from nicotine during the curing of tobacco, is metabolically activated by P450 enzymes. P450 2A6 is the primary hepatic catalyst of nicotine metabolism and also catalyzes NNK α-hydroxylation, albeit less efficiently. It was previously reported that P450 2B6 catalyzed nicotine 5′-oxidation. The studies presented here investigate the relative importance of P450 2B6 as a catalyst of nicotine 5′-oxidation and NNK α-hydroxylation by human liver microsomes (HLMs). Radioflow high-performance liquid chromatography analysis and tritiated substrates were used to monitor the products of nicotine and NNK metabolism. The primary product of P450 2B6-catalyzed nicotine metabolism was the Δ1′(5′) iminium ion. The only other metabolite detected was nornicotine, the product of methyl oxidation, formed at about one-fourth the rate of the Δ1′(5′) iminium ion. We determined that P450 2B6 was a much less efficient catalyst of nicotine 5′-oxidation than previously reported, with an estimated Km of 820 μM. In contrast, the Km of NNK α-hydroxylation was 33 μM. Experiments with P450 2A6- and P450 2B6-selective inhibitory antibodies did not support P450 2B6 as a significant catalyst of nicotine 5′-oxidation by HLMs, and it is unlikely that this enzyme contributes to nicotine metabolism in smokers who express P450 2A6. However, P450 2B6 contributed significantly to NNK metabolism in HLMs expressing both P450 2B6 and P450 2A6, suggesting a possible role for P450 2B6 in NNK metabolic activation.