Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na+ homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na+ accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na+ homeostasis, and citotoxicity were mimicked by stimulating protein kinase C (PKC) with 4β-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A1 abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na+ overload, and hepatocyte killing. Similarly, the addition of Na+ ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na+ accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na+ overload promoted by Na+-dependent acid buffering systems.