Hepatic complications are common in patients receiving total parental nutrition (TPN) and who have no underlying liver disease. In the present study we examined the hypothesis that endotoxin (LPS) or possibly TNF derived from the overgrowth of intestinal gram-negative bacteria is responsible for TPN-associated hepatic steatosis, and that bowel decontamination and specific anti-LPS activity of polymyxin B will reduce fatty infiltration of the liver during TPN. Forty-five male Sabra rats underwent jugular vein cannulation, were placed in metabolic cages, and were randomized into five groups. Group I was continuously infused with normal saline and allowed food ad lib, while group II-V were continuously infused with a TPN formula containing 4.25% amino acids and 25% dextrose for a total of 36 calories and 3.0 g protein per 100 g body weight/day. In addition, groups III-IV were also treated by oral polymyxin B while Groups IV and V received a combination of neomycin, metronidazole, and vancomycin (NMV). Thus, Group III received polymyxin B, Group IV received both polymyxin B and NMV, while Group V received NMV only. On Days 7-8 of the study, all animals were sacrificed and spontaneous production of TNF by peritoneal macrophages, bacterial translocation to mesenteric lymph nodes, culture of the cecum, and fat, triglyceride, and cholesterol contents of the liver were determined. All groups infused with TPN exhibited higher levels of total fat, triglycerides, and cholesterol compared to the free feeding control group (P < 0.001). Both groups receiving oral polymyxin exhibited a significant reduction in total liver fat (P < 0.02) and triglyceride (P < 0.001) levels compared to rats receiving TPN or TPN + NMV. The amount of gram-negative bacteria in the cecum of TPN-treated rats (Group II) was significantly higher than found in the free feeding rats (Group I). All antibiotic-treated groups had significantly lower gram-negative CFU than both the TPN and control groups due to bowel decontamination either by polymyxin B, NMV, or both. Simultaneously, spontaneous production of TNF by peritoneal macrophages of TPN-treated rats was significantly higher compared to control rats (P < 0.005) or those who received TPN with antimicrobial therapy (P < 0.03-0.005). In the present study both groups receiving polymyxin B exhibited reduced bacterial counts, reduced TNF production by peritoneal macrophages, representing the splanchnic reticuloendothelial system, and lower total fat and triglyceride levels in the livers of TPN rats. As the detrimental effects of LPS and the production of TNF in response to lipopolysaccaride were shown to be blocked by polymyxin B, this could well explain the protection afforded by polymyxin B against lipopolysaccharide-induced hepatic toxicity and steatosis, in addition to its very effective antibacterial activity in the gut, as demonstrated by the reduced gram-negative bacterial counts in the cecum of polymyxin B-treated rats.