Reductions in hepatic O₂ delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-B (NF-B) via generation of reactive O₂ species (ROS), the combination of these stimuli downregulates hepatic TNF- gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF- gene expression is transcriptionally mediated by reduced activation of NF-B. Buffer-perfused rat livers (n = 52) were studied over 180 min after intraportal infection at t = 0 with 10⁹ live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (PO₂ ~41 ± 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia (t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-B activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using ³²P-labeled oligonucleotides specific for NF-B. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-B nuclear translocation and TNF- bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O₂ consumption. XO inhibition reversed the hypoxic suppression of NF-B nuclear translocation and ameliorated decreases in cell-associated TNF-. Thus decreases in hepatic O₂ delivery reduce postbacteremic nuclear translocation of NF-B and hepatic TNF- biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.