Hepatic congestion occurs early in acetaminophen poisoning. This study examines whether acetaminophen is toxic to sinusoidal endothelial cells (SEC), which might lead to microcirculatory disruption. Acetaminophen toxicity was examined in vivo and in vitro in SEC and hepatocytes from C3H-HEN and Swiss Webster mice. In both strains, there was significantly more toxicity to SEC than to hepatocytes; in SEC from C3H-HEN mice, acetaminophen was directly toxic, but the presence of hepatocytes was required for toxicity to Swiss SEC. Acetaminophen, 750 mg/kg, by gavage caused toxicity with variability within and between strains, but all animals died between 3.5 and 6 hr with zone 3 hemorrhagic necrosis. Pretreatment of C3H-HEN SEC with aminobenzotriazole, a suicide inhibitor of P450, abolished toxicity. Baseline glutathione (GSH) levels were comparable, but a 12-hr incubation with acetaminophen decreased GSH by 60 and 8%, respectively, in C3H-HEN and Swiss SEC in single cell type culture. In co-culture, under conditions where Swiss SEC viability declined by 73%, hepatocyte viability and GSH only decreased by 21 and 20%, respectively. In conclusion, acetaminophen was toxic to SEC. It was directly toxic to SEC in one mouse strain and required hepatocyte activation in another strain. The lack of direct toxicity to Swiss SEC may be due to the lack of an activating P450 isozyme. Zone 3 hemorrhagic necrosis in vivo was comparable in both strains, despite differences in the pathways leading to SEC toxicity in vitro. We propose that toxicity to SEC may contribute to hepatic congestion in acetaminophen intoxication.