Activated macrophages convert L-arginine to citrulline and unstable nitrogen oxides that have cytotoxic properties. We recently have shown that the inhibition of protein synthesis in Kupffer cell (KC):hepatocyte (HC) coculture, following exposure to gram-negative bacterial endotoxin (lipopolysaccharide), is due to the metabolism of L-arginine by this cytotoxic pathway. Although this finding supports a role for activated KCs and the L-arginine-dependent mechanism in the HC dysfunction seen in sepsis, it and previous studies have failed to demonstrate direct damage to HCs by adjacent KCs. The current study was undertaken to determine if KCs exposed to lipopolysaccharide could directly damage HCs and, if so, whether the damage was dependent on the metabolism of L-arginine. By using the release of aspartate aminotransferase as a marker of HC damage, it was found that a significant aspartate aminotransferase release by KC:HC cocultures in response to lipopolysaccharide occurred only if L-arginine was present. In addition, requirements for significant aspartate aminotransferase release included KC:HC ratios of 7.5:1 or greater and L-arginine concentrations of 1 mmol or more. Although the KC-induced damage was mild, these results show that in vitro HC damage in KC:HC coculture does require the metabolism of L-arginine and supports a hypothesis that toxic L-arginine metabolites may contribute to liver cell damage in patients with sepsis.