The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated MeCG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 (MeC = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to MeCG sequences. In the present work, a stable isotope labeling HPLC-ESI+-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where MeC was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,-NH2-15N3-2-13C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI+-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2′-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3′-guanine bases within polypurine runs, while the formation of O 6-methyl-2′-deoxyguanosine and O6-[4-oxo-4-(3- pyridyl)but-1-yl]-2′-deoxyguanosine adducts was specifically preferred at the 3′-guanine base of 5′-GG and 5′-GGG sequences. In contrast, the presence of 5′-neighboring MeC inhibited O 6-guanine adduct formation. These results indicate that the N7- and O6-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.