Peroxiredoxins are a family of antioxidant enzymes conserved from bacteria to humans. In Saccharomyces cerevisiae, there exist five peroxiredoxins, among which Tsa2p shares striking homology with the well described Tsa1p but has not been extensively studied. Here we report on the functional characterization of yeast tsa2Delta mutants and the comparison of TSA1 with TSA2. The tsa2Delta and tsa1Delta tsa2Delta cells grew normally under aerobic conditions. However, the tsa1Delta tsa2Delta mutant yeast was more susceptible to oxidants than either tsa1Delta or tsa2Delta cells. Notably, the tsa1Delta tsa2Delta yeast was also hypersensitive to peroxynitrite and sodium nitroprusside. This phenotype was rescued by the expression of either the TSA1 or TSA2 gene. The demonstration of a peroxynitrite reductase activity of Tsa2p in vitro points to a pivotal role for peroxiredoxins in the protection against nitrosative stress. In yeast cells, Tsa1p and Tsa2p exhibited comparable antioxidant activity. While the basal expression level of TSA1 was significantly higher than that of TSA2, the transcription of TSA2 was stimulated more potently by various oxidants. In addition, TSA2 was activated in tsa1Delta cells in a Yap1p-dependent manner. Taken together, our findings implicate the cooperation of Tsa1p and Tsa2p in the cellular defense against reactive oxygen and nitrogen species.