The main N-alkylation products induced in DNA by methylating mutagens (7-methylguanine, 3-methyladenine, 3-methylguanine) are removed by excision repair involving, in the first step of the repair pathway, N-methylpurine-DNA glycosylase (MPG). To elucidate the significance of excision repair of N-alkylpurines in the defense of cells against alkylating agents we have modulated the efficiency of removal of N-methylpurines in Chinese hamster cells by transfecting them with the human MPG cDNA cloned into a mammalian expression vector. Although the stably transfected cells had a significantly higher capacity for removal of N-methylpurines from DNA, they did not gain protection against the cytotoxic and mutagenic effect of alkylating agents. The cells even responded more sensitively with respect to SCE formation. Here we show that the frequency of chromosomal aberrations induced by methyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine is significantly enhanced in the transfectants. Furthermore the transfectants showed a stronger inhibition of DNA replication and a higher yield of DNA breaks, as measured several hours after methylating agent exposure. The data suggest that overexpression of MPG causes an imbalance in the multi-step process of excision of N-methylpurines from DNA giving rise to a high yield of apurinic sites and/or gapped DNA that are intermediates in the formation of chromosomal aberrations, SCEs and the inhibition of replication in cells exposed to alkylating agents.