Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inactivated by peroxynitrite under biologically relevant conditions. The decrease of enzymatic activity followed an exponential function, and the concentration of peroxynitrite needed to inactivate 50% of 7 μM GAPDH (IC₅₀) was 17 μM. Hydroxyl radical scavengers did not protect GAPDH from inactivation, but molecules that react directly with peroxynitrite such as cysteine, glutathione, or methionine and the substrate, glyceraldehyde 3-phosphate, afforded significant protection. Assuming simple competition kinetics between scavengers and the enzyme, we estimated a second-order rate constant of (2.5 ± 0.5) × 10⁵M⁻¹s⁻¹at 25°C and pH 7.4 for the GAPDH tetramer. The loss of enzyme activity was accompanied by protein thiol oxidation (two thiols oxidized per subunit) with only one critical thiol responsible of enzyme inactivation. Indeed, the pH profile of inactivation was consistent with the reaction of GAPDH sulfhydryls (GAPDH-SH) with peroxynitrite. Peroxynitrite-inactivated GAPDH was resistant to arsenite reduction and only 15% recovered by 20 mM dithiothreitol, suggesting that GAPDH-SH has been mainly oxidized to sulfinic or sulfonic acid, with a minor proportion yielding a disulfide. On the other hand, under anaerobic conditions the peroxynitrite precursor, nitric oxide (^•NO), only slowly inactivated GAPDH with a rate constant of 11 M⁻¹s⁻¹. The remarkable reactivity of the critical thiol group in GAPDH (Cys-149) toward peroxynitrite, which is one order of magnitude higher than that of previously studied sulfhydryls, indicate that it may constitute a preferential intracellular target for peroxynitrite.