The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxicin vivo.Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P< 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P< 0.05). However, isoflurane was found to be less cytotoxic because lactate dehydrogenase leakage and morphology were not altered at concentrations as high as 2.35 mM and 5.5 mM in rat and human hepatocyte cultures, respectively. All of these observations show that halothane can be cytotoxic to adult hepatocytes maintained in standard culture conditions at concentrations much higher than those used in the clinical setting and that the degree of sensitivity of both cells is different.