The hepatotoxic effects of hyperthermic liver perfusion were investigated in male Fischer 344 rat livers. Perfusions were carried out at 37, 41, 42, 42.5, and 43Β°C for 2 hr. During the 2 hr, the perfusate was analyzed for activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), N-acetyl-/3-glucosaminidase (NAG), and glutathione (GSH), oxidized glutathione (GSSG), allantoin, and potassium. After perfusion, each liver was homogenized and analyzed for total xanthine oxidase (XO) activity, percentage type-D and type-0 XO, and total GSH content. Perfusate AST, LDH, NAG, and potassium levels were increased significantly with time and were significantly different in all hyperthermic perfusions from the 37Β°C perfusion values by the end of the perfusion. Perfusate GSH + GSSG levels were increased significantly in all hyperthermic perfusions after 60 min. Liver GSH levels were significantly lowered following perfusion at hyperthermic temperatures. There was a temperature-dependent increase in the percentage of XO in the type-0 form following perfusion at hyperthermic temperatures, which was strongly and positively correlated with the loss of hepatic GSH. These data support the hypothesis that hyperthermic toxicity to the liver is the result of oxidative stress brought about by conversion of XO to the type-0 form.