Peroxynitrite (ONOO^–) is a powerful oxidant exhibiting a wide array of tissue damaging effects including lipid peroxidation, change in activity of enzymes and ion channel via protein oxidation and nitration (Szabo, 2003). Here we examined peroxynitrite-induced K⁺ flux through the RBC membrane over a wide range of peroxynitrite concentrations (1-2000 μM) and its dependence on the time of peroxynitrite treatment (10-60 min). Peroxynitrite was synthesized from NaNO₂ and H₂O₂ and stabilized by alkali (Radi et al. 1991). Peroxynitrite was added as a bolus to the suspension of RBC (2-4% hematocrit), isolated from heparinised blood samples (from consenting volunteers with ethical permission), in buffer (NaCl or NaNO₃ 100 mM, Na₂HPO₄/Na₂HPO₄ 50 mM, pH 7.4 or 6.8, 37^oC). K⁺ influx was measured over 10 min at 37^oC using ⁸⁶Rb⁺ as a K⁺ congener, in the presence of ouabain (100 μM), bumetanide (1μM), and clotrimazole (10 μM) to obviate influx via the Na⁺-K⁺pump, the Na⁺-K⁺-2Cl^– cotransporter and the Gardos channel, respectively. Cl^–-dependent K⁺ influx was taken as a measure of K⁺-Cl^– cotransporter (KCC) activity. At peroxynitrite concentrations less than 300 μM, KCC is mainly activated with peak activity occurring at around 100 μM (KCC activity in control cells and cells after 100 μM peroxynitrite treatment was 0.56±1.46 mM (l cells)^-1 h^-1 and 8.24±1.04 mM (l cells)^-1 h^-1, respectively, p=0.00016). This value correlates well with concentrations previously shown to produce maximum effects on tyrosine kinase activity in human RBC stimulated by peroxynitrite (Mallozzi et al. 1999). At higher concentrations, peroxynitrite treatment of RBC leads to an increase in Cl^–-independent K⁺-influx with the peak of delta[K⁺]/delta[ONOO^–] at 220±48 μM (mean±S.E.M., n = 4), correlating with levels of methemoglobin formation and ATP depletion. The effect on both Cl^–-dependent and Cl^–-independent K⁺ fluxes induced by peroxynitirite was maximal following 30 min after peroxynitrite treatment. Neither NaNO₂, H₂O₂ or the products of peroxynitrite decomposition up to a concentration of 2 mM were able to induce an increase in Cl^–-independent K⁺ influx. Analysis of the concentration and time dependences of Cl^–-dependent and Cl^–-independent K⁺-influxes points to the presence of different sequential mechanisms underlying peroxynitrite actions on passive K⁺ transport of human RBC.