OBJECTIVE After small bowel resection, parenteral nutrition is often required to provide energy and nitrogen supplies and also to stimulate intestinal adaptation, despite the absence of glutamine in formulas. The aim of this study was to investigate the effect of nutrient supply route on fuel utilization by enterocytes. METHODS Rats received an intravenous or intragastric continuous infusion of an all-in-one glutamine-free formula. Sham-operated control rats were orally fed and received the same protein-caloric supplies as the other two groups. On day 7, the rats were killed in the fed state, blood samples were collected, and the jejunoileum was removed. Enterocytes were isolated. Aliquots of cell suspensions were incubated (30 minutes at 37 degrees C) in the presence of [14C]glucose and [14C]glutamine (2 mmol/L). Substrate utilization was determined by measuring metabolites and CO2 generated. RESULTS Intravenously fed rats showed mild hyperglycemia and marked hyperinsulinemia. Plasma glutamine levels were similar in the three groups. Intravenously fed rats showed a simultaneous increase in glutamine utilization and a decrease in glucose utilization compared with intragastrically fed and control rats, without parallel changes in glutaminase and hexokinase activities. The basolateral glucose transporter protein concentration was reduced in intravenously fed rat enterocytes. CONCLUSIONS The route of nutrient delivery influences fuel utilization by enterocytes.