Glycine has been shown to prevent hepatocyte death induced by anoxia and by several toxic agents. However, the mechanisms responsible for such a cytoprotective effect have not yet been entirely clarified. We have previously shown that an uncontrolled increase in intracellular Na+ is critical for hepatocyte killing induced by adenosine triphosphate (ATP) depletion. We herein report that protection by glycine (2 mmol/L) against cytotoxicity induced in isolated rat hepatocyte by potassium cyanide (KCN) or hypoxia was associated with the prevention of cytosolic Na+ accumulation. The addition of the Na+ ionophore, monensin, abolished the effects of glycine on both Na+ increase and cytotoxicity. Pretreating hepatocytes with the glycine-receptor antagonist, strychnine (1 mmol/L), similarly prevented Na+ overload and cell killing. Glycine at high concentrations and strychnine are known to block Cl- channels in many cell types. Consistently, we have observed that glycine and strychnine prevented the increase of intracellular Cl- levels caused by hypoxia or KCN. Incubation of hepatocytes in a Cl(-)-free medium, obtained by substituting chloride with membrane-impermeable gluconate, significantly reduced Na+ accumulation and cell killing triggered by hypoxia or KCN. Both these effects were abolished by the addition of monensin. The cytoprotective action exerted by hepatocyte incubation in the Cl(-)-free medium was, however, lost when membrane-permeable nitrate, which allowed Na+ accumulation, was used instead to replace chloride. Altogether, these results indicate that glycine inhibition of Cl- conductance protects against hepatocyte killing induced by KCN and hypoxia by interfering with intracellular Na+ accumulation triggered by ATP depletion.