Low arterial blood pH and sustained nitric oxide (NO) production are critical parameters in inflammatory events such as sepsis, and appropriate treatment is still under debate. Because the stability of nitrogen and oxygen intermediates is dependent on the surrounding pH, we investigated whether the relationship among NO, peroxynitrite (ONOO-), and reactive oxygen species production also depends on the pH value, particularly with respect to their effects on hepatocellular damage. Our studies demonstrate that the extracellular pH influences NO and hydroxyl radical (OH) production in hepatocytes. Acidification (pH 7.0) of the medium revealed a significant increase (P < 0.05) of OH-like radicals, enhanced hepatocellular damage, and a sharp drop in cellular glutathione (GSH) content compared with levels measured at physiological or alkaline pH conditions. Furthermore, inhibition of NO synthesis at all pH conditions resulted in decreased NO production and cellular GSH levels but a simultaneous increase of OH-like radicals and hepatocellular damage with a maximum seen at pH 7.0. Our results suggest that hepatocellular damage is in part regulated by the surrounding pH and that inhibition of NO synthesis at acidic conditions (e.g., in sepsis) leads to increased reactive oxygen-mediated cell injury.