In Escherichia coli, the repair of 3‐methyladenine (3MeA) DNA lesions prevents alkylation‐induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair‐deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag‐deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate‐induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation‐induced chromosome damage and cytotoxicity.