We have previously shown that the yeast Saccharomyces cerevisiae contains an antioxidant enzyme that can provide protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa enzyme was thus named thiol-specific antioxidant (TSA). We have now isolated and sequenced a yeast genomic DNA fragment that encodes TSA. Comparison of the predicted amino acid sequence of TSA with those of conventional antioxidant enzymes, including catalases, peroxidases, and superoxide dismutases, revealed no sequence homology. The 195-amino acid TSA sequence contains 2 cysteine residues. Southern blot analysis of petite yeast DNA, studies with protein synthesis inhibitors, and protein immunoblot analyses of cytosolic and mitochondrial proteins suggest that TSA is a cytosolic protein encoded by nuclear DNA (chromosome XIII). The yeast TSA gene was selectively disrupted by homologous recombination. The haploid tsa mutant was viable under air, suggesting that TSA is not essential for cell viability. The growth rates of the tsa mutant and wild-type strains were identical under anaerobic conditions. However, under aerobic conditions, especially in the presence of methyl viologen or a peroxide (t-butyl hydroperoxide or H2O2), the growth rate of the mutant was significantly less than that of wild-type cells. This result suggests that TSA is a physiologically important antioxidant.