Glucose metabolism and its hormonal regulation have been investigated in isolated enterocytes from rat small intestine. About 70% of the glucose consumed by the cells was transformed into lactate, 5% into pyruvate, and 4% into alanine. The remaining 20% was oxidized. Among several tested gastrointestinal peptides and hormones, only vasoactive intestinal peptide (VIP) was found to affect the metabolic fate of glucose. VIP (10(-7) M) induced a 40% inhibition of glucose oxidation without significant modification of either glucose uptake or production of lactate, pyruvate, and alanine. This acute inhibition was dose-dependent (Ki = 3.10(-11) M) and appeared to be dependent on the stimulation of cAMP production (K0.5 = 3.10(-9) M) since dibutyryl-cAMP and forskolin reproduced all the effects of VIP. Similar inhibition of cell respiration by VIP was observed when pyruvate, fructose, and dihydroxyacetone were used as substrates, while the oxidation of glutamine, ketone bodies, and octanoate was unaffected, suggesting that the peptide acts on pyruvate metabolism. The suppression of VIP effects by dichloroacetate (5 mM) and pyruvate (10 mM) and the significant decrease (18%) of the activity of the pyruvate dehydrogenase complex after incubation of the cells with the neuropeptide, support the hypothesis that the effects of VIP on glucose oxidation may occur through an inhibition of the pyruvate dehydrogenase complex. The total suppression of the inhibitory effects of VIP by sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of long-chain fatty acid oxidation, suggests that VIP did not affect the pyruvate dehydrogenase directly, but more probably acted through modifications of fatty acid oxidation.