Using a gastrostomy-fed (GF) rat infant "pup-in-a-cup" model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprived, non-Gln-treated group compared with mother-fed pups (P < 0.05). Gln blunted intestinal CINC mRNA (P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated approximately 13-fold compared with the mother-reared pups (P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide (P < 0.05). Gln but not Glu decreased plasma CINC peptide (P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% (P < 0.001) and 54% (P < 0.05), respectively. Intestinal and plasma TNF-alpha were increased in GF, LPS-treated pups (P < 0.01), and Gln and Glu both blunted this increase (P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.