The effect of tumor necrosis factor (TNF)-alpha and interleukin-1 (IL-1) alpha, which are thought to be principal mediators inducing homeostatic abnormalities during endotoxemia, were investigated on cultured hepatocytes in an attempt to understand their role in the pathogenesis of fulminant hepatitis. Both TNF and IL-1 had no direct cytotoxicity on cultured adult rat hepatocytes as assessed by their effects on protein synthesis and also cytosolic enzyme activity released into the culture medium in the presence of 5 mM D-galactosamine (Ga1N). However, IL-1 caused a dose-dependent inhibition of DNA synthesis in cultured adult rat hepatocytes. Moreover, the serum from TNF-treated rats, prepared after intravenous administration of TNF (5 X 10(4) U per rat), caused a significant increase of enzyme release into culture medium in contrast to control rat serum. The cytotoxicity disappeared when the serum from TNF-treated rats was pretreated by heating at 56 degrees C for 30 min, and was decreased by the addition of the protease inhibitor, aprotinin. In vivo, gabexate mesilate, a serine-type protease inhibitor, prevented GalN/TNF-induced fulminant hepatitis, whereas MX-1, an anti-complement agent, had no such effect. These results strongly suggest that IL-1 has a inhibitory effect on hepatocytes in terms of DNA synthesis and that TNF indirectly induces hepatocellular damage through the serine proteases which are possibly activated by the cytokine in vivo.