Ischemic preconditioning improves liver resistance to hypoxia and reduces reperfusion injury following transplantation. However, the intracellular signals that mediate the development of liver hypoxic preconditioning are largely unknown. We have investigated the signal pathway leading to preconditioning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned by 10-minute incubation under hypoxic conditions followed by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditioning reduced hepatocyte killing by hypoxia by about 35%. A similar protection was also obtained by preincubation with chloro-adenosine or with A2A-adenosine receptor agonist CGS21680, whereas A1-adenosine receptor agonist N-phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the development of preconditioning was blocked by A2-receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), but not by A1-receptor antagonist 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX). In either preconditioned or CGS21680-treated hepatocytes a selective activation of δ and ϵ protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric Gi protein or of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibitor PD98509 did not interfere with preconditioning that was instead blocked by p38 MAP kinase inhibitor SB203580. The direct activation of p38 MAPK by anisomycin A mimicked the protection against hypoxic injury given by preconditioning. Consistently, an increased phosphorylation of p38 MAPK was observed in preconditioned or CGS21680-treated hepatocytes, and this effect was abolished by PKC-blocker, chelerythrine. We propose that a signal pathway involving A2A-adenosine receptors, Gi-proteins, phospholypase C, δ- and ϵ-PKCs, and p38 MAPK, is responsible for the development of liver ischemic preconditioning.