O-Methylguanine-DNA-methyltransferase (MGMT) is a direct-reversal DNA repair protein that removes DNA adducts formed by alkylating mutagens found in tobacco smoke. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. However, their effect on the levels and types of genetic damage induced by specific environmental carcinogens remains to be fully elucidated. We developed two novel genotyping techniques and used them, in conjunction with the mutagen-sensitivity assay, to test the hypothesis that the L84F and I143V cSNPs in the MGMT gene confer increased sensitivity to genetic damage induced by the alkylating tobacco-specific nitrosamine carcinogen NNK. Lymphocytes from 114 healthy volunteers were exposed in vitro to NNK, and the genotoxic response was assessed by measuring chromosome aberration (CA) frequencies. A significant (P<0.02) increase in NNK-induced CA was observed in cells from individuals with the 84F polymorphism compared to cells from individuals homozygous for the referent L84 allele. A significant positive interaction between this cSNP and smoking, gender and age was observed (P<0.03). In subjects with the variant 143V allele, significantly higher levels of NNK-induced CA were observed in males and in young subjects (<43 years old) compared to subjects homozygous for the referent I143 allele (P<0.02). Individuals who inherited two cSNPs had significantly higher levels of NNK-induced CA compared to individuals with none or with one cSNP (P<0.002). These new data suggest that the 84F and 143V cSNPs may alter the function characteristics of the MGMT protein, resulting in suboptimal repair of genetic damage induced by NNK.