The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2'-deoxyguanosine (M(7)dG). M(7)dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E coli and Aag in humans. Structural studies of M(7)dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile M(7)dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2'-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in the DNA deprotection reaction. Duplex DNA containing 2'-fluoro-m(7)dG (FM(7)dG) cocrystallized with AlkA as a host-guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of FM(7)dG:C is nearly identical to that of G:C, and FM(7)dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical M(7)dG lesions.