Although administration of 100 mg galactosamine caused severe hepatic injury in C3H/HeN mice, splenectomy reduced the grade of this hepatotoxicity. However, this hepatic injury was scarcely detected in the endotoxin-resistant C3H/HeJ mice. In addition, in contrast to high lethality in C3H/HeN mice with a combined administration of galactosamine and endotoxin, splenectomy rendered C3H/HeN mice slightly resistant to this treatment. Further resistance was demonstrated in C3H/HeJ mice. In an attempt to clarify the role of endotoxin-responsive spleen cells in the pathogenesis of hepatic injury, we investigated galactosamine-induced hepatic injury by transfer of lipopolysaccharide-treated C3H/HeN or C3H/HeJ spleen cells. Both oxygen-derived free radical production and the proportion of macrophages in spleen cells were markedly enhanced in C3H/HeN mice after an intraperitoneal injection of lipopolysaccharide. Further increase in oxidative free radical production was found in the dish-adherent cells (macrophages). These enhancements were not demonstrated in lipopolysaccharide-treated C3H/HeJ spleen cells. Although hepatic injury was not demonstrated in both C3H/HeN and C3H/HeJ mice treated with 35 mg galactosamine alone, severe hepatotoxicity was found in these galactosamine-treated mice when they received lipopolysaccharide-activated C3H/HeN spleen cells, especially macrophages. Simultaneous administration of superoxide dismutase with the activated spleen cells reduced the grade of hepatic injury. On the other hand, hepatic injury was not demonstrated in the galactosamine-treated C3H/HeN or C3H/HeJ mice when they received lipopolysaccharide-treated C3H/HeJ spleen cells, although 3H-galactosamine incorporation into hepatocytes was nearly identical in both C3H/HeN and C3H/HeJ mice. These results suggest that oxidative free radicals of lipopolysaccharide-responsive macrophages could contribute to the pathogenesis of galactosamine-induced hepatic injury.