Peroxynitrite (ONOO⁻), a potent oxidizing and nitrating species, has been linked to covalent modifications of biomolecules in a number of pathological conditions. In S. cerevisiae, a model eukaryotic cell system, ONOO⁻ was found to be more potent than hydrogen peroxide in oxidizing thiols, inducing heat shock proteins (Hsp70) and enhancing the ubiquitination of proteins. As identified by microsequence analysis following immunoprecipitation with anti-nitrotyrosine antibodies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was especially susceptible to nitration by ONOO⁻ in yeast cells. The activity of this enzyme was strongly inhibited upon steady-state exposure of the cells to low doses of ONOO⁻ in yeast and in cultured rat astrocytes. Thus, ONOO⁻ is a potent stressor in yeast capable of inducing oxidative damage and protein nitration, with GAPDH being a preferential target protein that is efficiently inactivated.