Polymorphisms in genes of xenobiotic-metabolizing enzymes are largely responsible for interindividual differences in ability to activate and detoxify genotoxic agents and therefore may influence individual susceptibility to environmental cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), requires metabolic activation by cytochrome P450 (CYP) enzymes to generate DNA-reactive intermediates that induce mutations and cancer. In the current study, we investigated the role of the polymorphicCYP2E1andCYP2D6genes in the genotoxicity of NNK using the tandem-probe fluorescence in-situ hybridization (FISH) chromosome aberration assay as a marker. Our results, using whole blood cultures from 39 volunteers, indicated that NNK (0.12, 0.24 or 0.72 mM) induced a concentration-dependent increase in the frequency of chromosome aberration. The potential role ofCYP2E1andCYP2D6in NNK-induced genetic damage in cultured human lymphocytes was characterized using specific CYP inhibitors. Treatment of blood cultures with 25 μM diethyldithiocarbamate (DDC), a specificCYP2E1inhibitor, or 0.5 μM quinidine, a specificCYP2D6inhibitor, simultaneously with NNK, significantly decreased NNK-induced chromosome aberration. We also studied the role ofCYP2E1andCYP2D6allelic variants on NNK-induced chromosome aberration. Our results indicate that NNK induced a significantly higher level of chromosome aberration in cells with theCYP2E1 WT/*5Bgenotype compared to cells with theCYP2E1 WT/WT.In contrast, no difference in NNK-induced chromosome aberration was observed between cells with theCYP2D6extensive metabolizers compared to cells with theCYP2D6poor metabolizer genotypes. These results underscore the important role of polymorphic metabolizing genes in influencing the genotoxic responses to environmental mutagens and provide support to the reported findings linkingCYP2E1polymorphism to smoking-related lung cancer.