Lipopolysaccharide-binding protein (LBP) and CD14 play key intermediary roles in the activation of cells by endotoxin. As endotoxin has been postulated to participate in promoting pathological liver injury in alcoholic liver disease, we investigated the role of LBP and CD14 in alcoholic liver injury. Rats were fed intragastrically ethanol or dextrose and either medium-chain triglycerides, corn oil, or fish oil for 4 weeks. Kupffer cells, endothelial cells, and hepatocytes were isolated. LBP and CD14 mRNA levels were measured in liver and individual cell types. The highest levels of LBP and CD14 mRNA levels in the liver were found in the fish oil/ethanol group, which was also the group with the greatest degree of pathological injury and inflammation. CD14 mRNA levels were also significantly elevated in groups fed unsaturated fatty acids with dextrose. CD14 expression was localized to the Kupffer cells and LBP expression to the hepatocytes. Expression of CD14 mRNA was also found in nonmyeloid cells in the two experimental groups (fish oil/ethanol and corn oil/ethanol) that had liver necrosis and inflammation. Our results suggest that enhanced LBP and CD14 expression correlates with the presence of pathological liver injury in alcoholic liver injury. Furthermore, unsaturated fatty acids may prime cells to respond to endotoxin by enhancing CD14 expression.