Peroxynitrite (ONOO) is a reactive nitrogen species produced when nitric oxide (NO) and superoxide react. In vivo studies suggest that reactive oxygen species and perhaps peroxynitrite can influence Na,K-ATPase function. However, the direct effects of peroxynitrite on Na,K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na, K - ATPase activity with an IC₅₀ of 107 ± 9 uM. To mimic cellular/physiological production of peroxynitrite a syringe pump was used to slowly release ( 0.85 uM / second) peroxynitrite. This treatment was similarly effective at inhibiting Na,K-ATPase activity as a single bolus addition of equal cumulative concentration. Peroxynitrite treatment produced 3-nitrotyrosine residues on the , and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration was unable to blunt peroxynitrite induced ATPase inhibition suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite lead to a decrease in iodoacetamidofluorescein labeling implying cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na⁺ and low Mg ATP during peroxynitrite treatment increased the IC₅₀ to 145 ± 10 uM while the presence of K⁺ and low Mg ATP increased the IC₅₀ to 255 ± 13 uM. This result suggests that the E-PNa conformation of the pump is slightly more sensitive to peroxynitrite than the E (K) conformation. Taken together these results show that peroxynitrite is a potent inhibitor of Na,K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.