We previously cloned a DNA fragment from Saccharomyces cerevisiae that suppressed the alkylation sensitivity of Escherichia coli glycosylase deficient mutants and we showed that it apparently contained a gene for 3-methyl-adenine DNA glycosylase (MAG). Here we establish the identity of the MAG gene by sequence analysis and describe its in vivo function and expression in yeast cells. The MAG DNA glycosylase specifically protects yeast cells against the killing effects of alkylating agents. It does not protect cells against mutation; indeed, it appears to generate mutations which presumably result from those apurinic sites produced by the glycosylase that escape further repair. The MAG gene, which we mapped to chromosome V, is not allelic with any of the RAD genes and appears to be allelic to the unmapped MMS-5 gene. From its sequence the MAG glycosylase is predicted to contain 296 amino acids and have a molecular weight of 34,293 daltons. A 137 amino acid stretch of the MAG glycosylase displays 27.0% identity and 63.5% similarity with the E. coli AlkA glycosylase. Transcription of the MAG gene, like that of the E. coli alkA gene, is greatly increased when yeast cells are exposed to relatively non-toxic levels of alkylating agents.