Yeast and mammalian cells contain a 25 kDa enzyme that protects cellular components against oxidative damage from a system capable of generating reactive sulfur species, but not from a system that generates only reactive oxygen species. Yeast and rat cDNAs corresponding to this thiol-specific antioxidant (TSA) have been cloned and sequenced. Rat TSA is 65.3% identical and 76.2% similar to yeast TSA in amino acid sequence. A search of the GenBank database revealed 12 additional TSA-like proteins, which show sequence identity to rat TSA ranging from 31 to 76%. Except for the AhpC protein identified in Salmonella typhimurium, none of the TSA-like proteins is associated with known cellular functions. AhpC, which exhibits approximately 40% sequence identity to TSA, has been proposed to be a catalytic component of alkyl hydroperoxide reductase. Alignment of rat and yeast TSA with the TSA-like sequences revealed two conserved cysteine residues, one conserved in all 14 sequences and the other in 12 sequences. The most conserved cysteine is located in a well-conserved motif of (hydrophobic residue)6-Pro-non-conserved-residue-Asp-Phe-Thr-Phe-Val-Cys-Pro-Thr-Glu- hydrophobic residue. These results suggest that the TSA-like proteins of previously unknown function may represent a widely distributed family of antioxidants with functions similar to those of TSA and AhpC.