This study determined splanchnic extraction of phenylalanine at two intakes of threonine. Six Thoroughbred mares were supplemented with isonitrogenous amounts of either threonine or glutamate. Dietary threonine intakes were 119 (+Thr) and 58 (Basal) mg/kg/day, respectively. Each horse received each diet twice and each was studied once with an oral and once with an intravenous (IV) infusion of [1-(13)C]phenylalanine. A 2-h primed, constant IV infusion of [(13)C]sodium bicarbonate and a 4-h primed, constant infusion of [1-(13)C]phenylalanine, either orally or IV, were used to measure isotopic enrichments. Phenylalanine kinetics were not affected by diet (P > 0.05). Values for the splanchnic extraction of phenylalanine were 26 ± 5% and 27 ± 3% for the +Thr and Basal supplemented diets, respectively. These values will improve the accuracy of future equine indicator amino acid oxidation studies.