This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells. Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin. Male Sprague-Dawley rats received a nonlethal (“lowdose”) intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a “large dose” of endotoxin (10 mg/kg). Three hours after LPS challenge, the livers were perfused, and superoxide release was determined. Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro. There was a low rate (0.14 ± 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously. Control livers generated less than 0.08 nmol superoxide. A high rate (1.3 ± 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS. This was attenuated to 0.7 ± 0.04 nmol/min/g by an injection of low-dose LPS before challenge. This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS. Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan. Pretreatment of rats with the low dose of LPS 48 hours before the large dose attenuated the spontaneous and primed release of superoxide by isolated nonparenchymal cells. Serum transaminase activities were also reduced in these LPS-tolerant rats. The suppressed oxygen-derived radical formation by the liver and nonparenchymal cells during acute LPS tolerance was associated with down-regulation of tumor necrosis factor (TNF) release in vivo. TNF has been shown to stimulate and prime neutrophils and Kupffer cells for increased respiratory burst. These results suggest that suppression of free radical formation may contribute to the amelioration of hepatic injury during acute endotoxin tolerance.