This study tested the hypothesis that L-glutamine (Gln) or L-alanyl-L-glutamine (Ala-Gln) prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Enterocytes of neonatal pigs rapidly hydrolyzed Ala-Gln and utilized Gln. To determine whether Gln or Ala-Gln has a cytoprotective effect, IPEC-1 cells were cultured for 24 h in Gln-free Dulbecco's modified Eagle's-F12 Ham medium containing 0, 0.5, 2.0 or 5.0 mM Gln or Ala-Gln, and 0, 0.5 mM H(2)O(2) or 30 ng/ml lipopolysaccharide (LPS). Without Gln or Ala-Gln, H(2)O(2)- or LPS-treated cells exhibited almost complete death. Gln or Ala-Gln at 0.5, 2 and 5 mM dose-dependently reduced H(2)O(2)- or LPS-induced cell death by 14, 54 and 95%, respectively, whereas D: -glutamine, alanine, glutamate, ornithine, proline, glucosamine or nucleosides had no effect. To evaluate the effectiveness of Gln or Ala-Gln in vivo, 7-day-old piglets received one-week oral administration of Gln or Ala-Gln (3.42 mmol/kg body weight) twice daily and then a single intraperitoneal injection of LPS (0.1 mg/kg body weight); piglets were euthanized in 24 and 48 h to analyze intestinal apoptotic proteins and morphology. Administration of Gln or Ala-Gln to LPS-challenged piglets increased Gln concentrations in small-intestinal lumen and plasma, reduced intestinal expression of Toll-like receptor-4, active caspase-3 and NFkB, ameliorated intestinal injury, decreased rectal temperature, and enhanced growth performance. These results demonstrate a protective effect of Gln or Ala-Gln against H(2)O(2)- or LPS-induced enterocyte death. The findings support addition of Gln or Ala-Gln to current Gln-free pediatric amino acid solutions to prevent intestinal oxidative injury and inflammatory disease in neonates.