To quantitate glutamine kinetics in premature infants and determine whether glutamine affects leucine metabolism, 11 very low birth weight (<1250 g) neonates received 4-h i.v. infusions of L-[2H3]leucine and L-[13C5]glutamine, along with orogastric infusion of L-[1-13C]leucine and L-[1-13C]glutamine on the 10th d of life and in the fed state. Patients were receiving parenteral nutrition and were randomized to receive either hypocaloric, enteral preterm formula alone(controls;n= 5), or glutamine (0.2 g·kg-1·d-1on the day of the study) supplemented formula (GLN;n= 6). The rates of appearance (Ra) of leucine and glutamine, and their rates of splanchnic extraction were determined from isotopic enrichments in plasma at steady state. Leucine release from protein breakdown did not differ between groups (123 ± 51versus162 ± 94 μmol·kg-1h-1in the controls and GLN group, respectively). Glutaminede novosynthesis accounted for >80% of overall glutamineRa, and was similar in both groups (626 ± 177versus525 ± 86μmol·kg-1·h-1; NS); 46 ± 16% and 53± 31% of the enteral glutamine underwent first-pass splanchnic extraction in the controls and GLN group, respectively. These findings indicate that the pathways of glutaminede novosynthesis and glutamine utilization in the splanchnic bed are functional in very low birth weight humans by the 10th d of life. Glutamine supplementation provided at low doses on a hypocaloric regimen results in no apparent differences in flux of glutamine or leucine.Abbreviations: VLBW,very low birth weight;PN,parenteral nutrition;GCMS,gas chromatography-mass spectrometry;OG,orogastric;KIC,α-ketoisocaproic acid;Ra,rate of appearance