Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1‐methyladenine (1meA) and 3‐methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene‐targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2‐deficient mice are unable to remove methyl methane sulfate (MMS)‐induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double‐stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.