This study was designed to test the hypothesis that endogenous arginine synthesis plays an important role in maintaining arginine homeostasis in neonatal pigs. Gabaculine was used as a suicide inhibitor of ornithine aminotransferase to decrease the intestinal conversion of glutamine-derived pyrroline-5-carboxylate (P-5-C) into ornithine, the precursor of both citrulline and arginine. Four-day-old suckling pigs received oral administration of 0.0 or 0.83 mg gabaculine/kg body wt every 4 h during a 12-h period from 6 A.M. to 6 P.M. Blood was collected from piglet's jugular vein at 6 A.M. and 6 P.M. after a 2-h isolation from sows. Gabaculine treatment decreased plasma concentrations of ornithine, citrulline, and arginine by 59, 52, and 76%, respectively, and increased those of glutamine and proline by 74 and 220%, respectively. The gabaculine treatment also increased plasma concentrations of leucine, taurine, and ammonia by 29, 42, and 20%, respectively. There were no differences in intramuscular concentrations of amino acids between control and gabaculine-treated pigs. Because P-5-C synthase (the enzyme required for synthesis of P-5-C from glutamate) was almost exclusively located in enterocytes of 4-day-old pigs, our data suggest that the intestinal production of citrulline plays an important role in endogenous synthesis of arginine and its homeostasis in neonatal pigs.