The biological targets of peroxynitrite toxicity include wide array of biomolecules. Although several enzymes are found to be important components of cellular defense against peroxynitrite, the complete scenario is not totally understood. Yeast flavohemoglobin (YHB) and glutathione-dependent formaldehyde dehydrogenase (GS-FDH) confers resistance against nitric oxide and related reactive nitrogen species. In the present study, when subtoxic dose of peroxynitrite was applied to wild type, Deltayhb1 and Deltasfa1 strains of Saccharomyces cerevisiae, induction of cytosolic catalase was found at activity as well as gene expression level in mutants but not in wild type. Such induction was not due to intracellular reactive oxygen species (ROS) formation. Our in vitro studies confirmed the role of catalase in protection against peroxynitrite-mediated oxidation and nitration and also in peroxynitrite catabolism. This report is first of its kind regarding the novel role of catalase in peroxynitrite detoxification in Deltayhb1 and Deltasfa1 strains of S. cerevisiae.