We recently described the antibacterial activity of a murine hepatocyte cell line stimulated with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and lipopolysaccharide (LPS) against intracellular Salmonella organisms. Here we show for the first time the existence of basal antibacterial activity in cultured hepatocyte cell lines. Thus treatment of resting and stimulated hepatocytes with catalase or superoxide dismutase increased bacterial number recovered per monolayer, which suggests that the mechanism involved with antibacterial activity of hepatocytes is mediated by reactive oxygen species (ROS). Also, the capacity of these cell lines to generate intracellular peroxides under resting and stimulated conditions was investigated. This revealed that IL-1 and LPS did not induce any increase in the amount of intracellular peroxides by themselves, but they primed IFN-gamma for maximal induction of peroxides. The intracellular amount of peroxides was highly increased on stimulation with IFN-gamma, IL-1, and LPS, and it was strongly inhibited by catalase. This explains that the mechanism whereby this enzyme inhibits antibacterial activity takes place by decreasing the intracellular pool of peroxides. In turn, experiments performed in the presence of several inhibitors of metabolic pathways involved in ROS generation suggested that cyclo-oxygenase are a source of these species in hepatocyte cell lines. These results attribute a prominent role to the generation of peroxides as effector molecules of antibacterial activity in hepatocyte cell lines. Thus these cells displayed a moderate basal level, which increased on stimulation with proinflammatory cytokines such as IFN-gamma, IL-1, and bacterial products such as LPS. Finally, it has been also shown for the first time that IFN-gamma stimulation induces production of peroxides in human and murine hepatocyte cell lines.