Human polymorphonuclear neutrophils, when exposed to soluble or particulate stimuli, can destroy various types of cells. The aim of this study was to investigate their toxicity against hepatocytes. Human polymorphonuclear neutrophils were incubated in basal conditions and after stimulation with 5 mg per ml opsonized zymosan in the presence of rat hepatocytes isolated by collagenase digestion. Cytotoxicity was quantified by the percentage of ALT activity released by hepatocytes in culture medium. Whereas unstimulated neutrophils exhibited only minor effects, opsonized zymosan-stimulated neutrophils induced, after 16 hr incubation, a 24.0 ± 4.1% (mean ± 1 S.E.) ALT activity release at a neutrophil/hepatocyte ratio of 5, and a 51.7 ± 6.8% ALT activity release at a ratio of 20. At this ratio of 20, the ALT activity release was 9.0% at 1 hr and 24.0% at 4 hr. Three proteinase inhibitors (i.e., soybean trypsin inhibitor, α1-proteinase inhibitor and fetal calf serum) decrease cytotoxicity by 78, 76 and 78%, respectively. The protective effect of proteinase inhibitors was not due to a nonspecific effect of proteins, since bovine serum albumin did not decrease the toxicity of stimulated polymorphonuclear cells. The supernatant of stimulated neutrophils was also found to be toxic against hepatocytes, and again, this effect was inhibited by soybean trypsin inhibitor, α1-proteinase inhibitor and fetal calf serum. Finally, the role of proteinases was supported by the demonstration of a cytotoxic effect of two purified proteinases: porcine pancreatic elastase and human neutrophil cathepsin G. The toxicity of these proteinases was also markedly reduced by the specific inhibitors used in the study. These results show that human polymorphonuclear neutrophils stimulated with opsonized zymosan may destroy hepatocytes through the release of proteinases. This might have important path-ophysiological implications, especially in alcoholic hepatitis in which polymorphonuclear neutrophils regularly infiltrate the liver and could therefore participate in the hepatocellular injury.