We studied the absorption of enteral glutamate and phenylalanine using isotopic tracer and arteriovenous difference techniques. Six piglets, implanted with portal, carotid, and gastric catheters and an ultrasonic portal flow probe received a 6-h intragastric infusion of [U-13C] glutamate and [2H] phenylalanine, with a high-protein diet offered one time each hour. Amino acid concentrations and the isotopic enrichments of all mass isotopomers of glutamate, glutamine, and phenylalanine were measured in portal and arterial blood over the last hour. There was significant (P<0.025) net absorption of the indispensable amino acids as well as arginine, proline, serine, and alanine. There was no portal uptake of glutamate, aspartate, and glycine, and arterial glutamine was removed by the portal drained viscera (P<0.05). At isotopic steady state, 72% of the [2H] phenylalanine but only 5% of the [U-13C] glutamate tracer appeared in the portal blood. We conclude that, in fed infant pigs, the gut metabolizes virtually all of the enteral glutamate during absorption. Therefore, glutamate and glutamine in the body as a whole must derive almost entirely from synthesis de novo.