The purpose of the present study was to determine whether the lipopolysaccharide (LPS)-induced increase in glucose uptake was a generalized response along the length of the entire gastrointestinal (GI) tract and to assess the relative contributions of the mucosa and muscularis. The putative roles of tumor necrosis factor (TNF) and blood flow in the metabolic response of the intestine to LPS were also examined. In vivo glucose uptake (Rg) was determined for various segments of the GI tract under basal postabsorptive conditions and 3 h after intravenous injection of Escherichia coli LPS using 2-[14C]deoxyglucose. At this time, LPS-treated rats were euglycemic, and Rg was elevated in all sections of the GI tract (52-96%). In control animals, mucosal Rg accounted for 79% of the glucose uptake by the entire small intestine; LPS increased Rg in both the mucosa and muscularis and did not alter this relationship. The LPS-induced increase in intestinal Rg was not attenuated by pretreatment with TNF antibody. Cardiac output (CO) and intestinal blood flow, assessed using radiolabeled microspheres, were not different from control values 3 h after LPS. Blood flow to the muscularis was increased (120%) in all sections of the small intestine from LPS-treated rats. These results indicate that 3 h after a low dose of LPS, glucose uptake by the entire length of the GI tract was elevated, and the majority of increase was due to enhanced uptake by the mucosa, which was blood flow independent. Furthermore, the increased Rg was not dependent on elevations in plasma glucose, insulin, or TNF levels. However, an increased blood flow to the muscularis was associated with an elevated Rg in that region.