Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O(6)-POB-dG) lesions. If not repaired, O(6)-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O(6)-POB-dG can be directly repaired by O(6)-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O(6)-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of O(6)-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O(6)-POB-dG adducts within K-ras and p53 gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI(+)-MS/MS analysis of O(6)-POB-dG remaining in DNA over time. We found that the second-order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences) but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of O(6)-POB-dG was 2-7 times slower than that of O(6)-Me-dG adducts. To evaluate the contribution of AGT to O(6)-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O(6)-POB-dG adduct repair over time was monitored by HPLC-ESI(+)-MS/MS. We found that HBEC cells were capable of removing O(6)-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (O(6)-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of O(6)-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer.