Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, aSaccharomyces cerevisiae sod1Δ mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1H46C) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3Δ andgpx1Δ/gpx2Δ/gpx3Δ) and for the repair of DNA oxidation (ogg1Δ andrad30Δ/ogg1Δ) were tested, but were found not to be Cr-sensitive. In contrast,S. cerevisiae msraΔ (mxr1Δ) andmsrbΔ (ycl033cΔ) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity ofsod1Δ cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed ∼20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity inS. cerevisiae, which primarily involves oxidative damage to cellular proteins.