The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties. However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species. To elucidate the response of eukaryotic cells to 4-NQO-induced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen. One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation. A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated. Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate. Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair. These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.