The DNA repair protein, O6-alkylguanine DNAalkyltransferase (AGT), specifically recognizes and removes O6-alkyl substituents on guanine, restoring normal guanine and preventing mutagenesis. When AGT binds O6-alkyl-deoxyguanosine (O6-alkyl-dG) containing DNA, the protein interacts with the 1-, N2-, and 7- positions of the guanine lesion. The repair of O6-alkyl-dG lesions is a multi-step process. Following AGT binding to DNA, the adducted nucleotide is flipped into the active site of the protein, and the O6-alkyl substituent is displaced via an SN2-type mechanism, restoring normal guanine and producing alkylated protein. The rates of AGT repair are influenced by DNA sequence context, secondary structure, and alkyl group identity. The relative rates of AGT-mediated repair of O6-alkyl-dG lesions are benzyl > methyl > ethyl ≫ 2-hydroxyethyl >4-(3-pyridyl)-4-oxobutyl. The differences in rates of repair between different alkyl groups and different sequence contexts are not a result of difference in AGT binding and kinetics of nucleotide flipping, since these reaction steps are very fast and are unaffected by DNA sequence. The rate of alkyl transfer is the slowest forward step in the repair of O6-Me-dG and appears to be dependent on the alkyl group identity and is influenced by the local sequence context. AGT repair of O6-alkyl-dG lesions is essential for maintenance of genome integrity, and slow repair of these lesions in specific DNA sequences may contribute to the mutational spectra observed in human cancer.