Furan is a potent rodent hepatotoxicant and carcinogen. The present study was done to examine the effects of furan on hepatic energy metabolism both in vivo and in vitro in male F-344 rats. Furan produced concentration- and incubation time-dependent irreversible reductions in ATP in freshly isolated F-344 rat hepatocytes. Furan-mediated depletion of ATP occurred prior to cell death and was prevented by including 1-phenylimidazole, a cytochrome P450 inhibitor, in the suspensions. Male F-344 rats were treated with furan (0-30 mg/kg, po) and killed 24 hr later to prepare hepatic mitochondria. Furan produced dose-dependent increases in state 4 respiration and ATPase activity. Both of these changes were prevented by 1-phenylimidazole cotreatment. In a separate series of experiments, mitochondria were prepared from isolated rat hepatocytes following incubation with furan (2-100 microM) for 1-4 hr. Furan produced incubation time- and concentration-dependent increases in state 4 respiration and ATPase activity. Furan-mediated mitochondrial changes were prevented by adding 1-phenylimidazole to the hepatocyte suspensions. These results indicate that the ene-dialdehyde metabolite of furan uncouples hepatic oxidative phosphorylation in vivo and in vitro. In vitro studies using an isolated hepatocyte suspension/culture system demonstrated that the concentration response for furan-mediated mitochondrial changes in suspension corresponded with the concentration responses for cell death after 24 hr. Including 1-phenylimidazole or oligomycin plus fructose in hepatocyte suspensions prevented furan-induced cell death after 24 hr in culture. The results of this study indicate that furan-induced uncoupling of oxidative phosphorylation is an early, critical event in cytolethality both in vivo and in vitro.