Bleomycin is an antitumor drug that damages DNA via a free radical-dependent mechanism, and yeast mutants defective in DNA repair are hypersensitive to the drug. To identify possible pathways that may contribute to bleomycin resistance in yeast, we characterized a panel of bleomycin-sensitive mutants that were previously isolated by insertion mutagenesis using the transposon miniTn3::Leu2::LacZ::AMP( R). One of these mutants harbored a single insertion in the SLG1 gene, which encodes a cell membrane protein that senses cell wall stress, and functions to maintain cell wall function by activating the protein kinase C signaling pathway. Deletion of the SLG1 gene in parental strains caused hypersensitivity to bleomycin, and this correlated with an accumulation of damaged DNA. A plasmid that expresses the native SLG1 gene or that increases PKC1 gene dosage restored bleomycin resistance to the slg1Delta mutant. Two-dimensional gel electrophoresis revealed that exposure to bleomycin triggered the expression of certain proteins, presumably to maintain cell wall function, in a Slg1-dependent manner. In addition, mutants lacking cell wall function were found to be hypersensitive to bleomycin. We conclude that mutants deficient in proteins that maintain cell wall function are severely compromised in their ability to limit bleomycin entry into the cell. Therefore, these mutants are burdened with increased genotoxicity upon exposure to bleomycin in the medium. Our results show that major mechanisms other than DNA repair are operating in yeast to mediate bleomycin resistance.