We performed this study to determine whether perfused isolated human and rat hepatocytes have different sensitivities to anoxia-reoxygenation injury. Oxygen free radicals were detected by lucigenin-enhanced chemiluminescence. Lipid peroxidation was assessed by measuring malondialdehyde release. Cell injury was evaluated by measuring lactate dehydrogenase release and trypan blue uptake. During the control period, lucigenin-enhanced chemiluminescence, malondialdehyde and lactate dehydrogenase release and trypan blue uptake were similar in rat and human hepatocytes. During 3.5 hr of anoxia, lucigeninenhanced chemiluminescence decreased to background levels and malondialdehyde release remained constant in both groups. In contrast, lactate dehydrogenase release increased eightfold in rat hepatocytes but only threefold in human hepatocytes. With reoxygenation after 2.5 hr of anoxia, in rat hepatocytes lucigenin-enhanced chemiluminescence increased 13-fold within 15 min and then declined toward control levels. Malondialdehyde release doubled after 1 hr of reoxygenation. The rate of lactate dehydrogenase release increased to a level almost twice that observed in cells kept continuously anoxic. In contrast, with human hepatocytes lucigenin-enhanced chemiluminescence increased only fourfold, whereas malondialdehyde and lactate dehydrogenase releases did not differ significantly from those levels measured in cells perfused continuously under anoxic conditions. At the end of the experiment, the increase in trypan blue uptake was significantly greater with rat hepatocytes