The purposes of this study were to determine the pH dependence of lethal endothelial cell injury during oxidative stress and the pH dependence of those cellular mechanisms proposed to result in endothelial cell killing. Oxidative stress was produced in rat liver sinusoidal endothelial cells with H2O2 (5 mmol/L). Cell survival was dependent on the extracellular pH. Indeed, after 180 min of incubation with H2O2, cell survival was only 27% at pH 7.4, 45% at pH 6.8 (p less than 0.05) and 62% at pH 6.4 (p less than 0.05). Despite improved cell survival at pH 6.4 compared with pH 7.4, the magnitude of ATP hydrolysis and glutathione depletion was similar. In contrast to cell survival, lipid peroxidation as measured by malondialdehyde generation was increased twofold at pH 6.4 compared with pH 7.4. A rapid and profound loss of the mitochondrial membrane potential occurred during oxidative stress at pH 7.4 that was delayed at pH 6.4 (0.3% vs. 20% of the initial value at 30 min, p less than 0.0001). After 60 min of incubation with H2O2, NAD(P)H oxidation was greater at pH 7.4 than at pH 6.4 (100% vs. 64%, p less than 0.05). The results indicate that the protective effect of acidosis against cell death during oxidative stress is associated with the inhibition of NAD(P)H oxidation and delayed loss of the mitochondrial membrane potential. Acidosis appears to maintain organelle and cell integrity during oxidative stress by influencing the redox status of NAD(P)H.