O6-Methyl-2′-deoxyguanosine (O6-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK). O6-Me-dG is directly repaired by the specialized DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), which transfers the O6-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O6-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/ pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O6-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5′-G1TA G2TT G 3G4A G5CT G6G7T G 8G9C G10TA G11G12C AAG13 AG14T-3′, where G5, G6, G7, G8, G9, G10, or G11 was replaced with O6-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G → A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O6-Me-dG- containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of C6-Me-G remaining in DNA. The relative extent of demethylation for O6-Me-dG adducts located at G5, G6, G 7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O6-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5′-G6G7T-3′) were similar (1.4 × 107 M-1 s-1 vs 7.4 × 10 6 M-1 s-1, respectively), suggesting that O6-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.