NNK is a potent carcinogen, generated during tobacco processing and smoking that requires metabolic activation by various cytochrome P450 (CYP450) enzymes in order to exert its carcinogenic effect. In the present study we investigate the role of polymorphic CYP2D6 variants in activating NNK to induce sister chromatid exchanges (SCEs) and chromosome aberration (CA) using fluorescence in situ hybridization (FISH) in cultured human lymphocytes. Our results indicate that human lymphocytes are capable of activating NNK to induce SCE and CA in a concentration-dependent manner (25-300 µg NNK/ml). Furthermore, our data indicate a significant difference (P ≤ 0.01) in NNK-induced SCE between lymphocytes from CYP2D6 extensive metabolizers (EMs) compared to poor metabolizers (PMs). In addition, we observed a difference in NNK-induced SCE between homozygous and heterozygous EM CYP2D6 individuals. Homozygous EM individuals showed a mean of 1.7 fold increase in SCEs compared to heterozygous individuals at all concentrations tested. Using the sensitive FISH assay, we observed that the simultaneous addition of quinidine (0.5 µM), a specific CYP2D6 inhibitor, with NNK (50 µg/ml) protected the cells from NNK-induced CA (30% reduction in CA). Taken together, our data indicate that allelic variants of CYP2D6 gene can significantly affect the metabolic activation of NNK to induce genetic damage. These results provide additional evidence that CYP2D6 is an important susceptibility factor for cigarette-smoke related cancers and underscore the important role of this polymorphic gene in influencing the genotoxic responses to environmental mutagens.