The predominant adduct produced by both endogenous and exogenous methylating agents is 7-methylguanine(m7G). Most studies on the repair of m7G reported so far used methylated DNA as substrates which contained other unintended lesions. In the presented study, DNA substrates containing m7G as unique lesions were prepared by DNA polymerase reactions. Using these substrates, damage recognition of E. coli 3-methyladenine DNA glycosylase II (AlkA) was analyzed. The obtained results suggested that the repair rate of m7G by AlkA was affected by the flanking sequence context of the lesion.