This study was directed at the role of tolerance to endotoxin (lipopolysaccharide, LPS) and ethanol (EtOH) intoxication in modulating hepatic nitric oxide (NO) production, and the demonstration of gender differences. Previous studies demonstrated that tolerance to either LPS or EtOH was associated with reduced hepatic production of superoxide anions. We now tested the hypothesis that the reduced hepatic production of superoxide anions during tolerance to LPS and the altered response to EtOH are accompanied by increased sensitivity of hepatic NO release to stimulation. Age-matched male and female Sprague-Dawley rats were made tolerant to LPS by an i.v. injection of LPS (0.5 or 0.45 mg/kg) 2 days prior to an in vivo EtOH infusion for 3 h (LPS-EtOH group). Control groups were saline-pretreated, saline-infused; saline-pretreated, EtOH-infused; and LPS-pretreated, saline-infused. At the end of the infusion, isolated hepatocytes, Kupffer, and sinusoidal endothelial cells were cultured for 20 h for subsequent measurement of basal (spontaneous) and in vitro-stimulated nitrite release. LPS-tolerance resulted in significantly enhanced stimulated NO production by hepatocytes and Kupffer cells in both male and female rats. EtOH abolished this priming effect in hepatocytes from male, but not from female rats. The priming effect was markedly diminished by EtOH in Kupffer cells of female rats only. LPS-tolerance increased NO production by stimulated endothelial cells of males, and decreased NO production by cells of females. EtOH infusion did not influence NO production by endothelial cells from male rats and it reversed the LPS-tolerance-induced inhibition in females. These data demonstrate that modulation by LPS-tolerance of hepatic NO release in EtOH-treated rats leads to enhanced stimulated NO production, while hepatic superoxide anion release was previously shown to be reduced within the same time frame. Since NO is able to scavenge superoxide, the LPS-tolerance-induced alterations in the EtOH effects on NO production may have a potential significance in modulating - in a time-dependent manner - oxidative injury associated with LPS and acute EtOH intake.