BACKGROUND: Glutamine exhibits numerous beneficial effects in experimental and clinical studies. It has been suggested that these effects may be partly mediated by the conversion of glutamine into citrulline and arginine. The intestinal metabolism of glutamine appears to be crucial in this pathway. The present study was designed to establish the effect of the feeding route, enteral or parenteral, on the conversion of exogenously administered glutamine into citrulline and arginine at an organ level in humans, with a focus on gut metabolism. METHODS: Sixteen patients undergoing upper gastrointestinal surgery received an IV or enteral (EN) infusion of l-[2-(15)N]glutamine. Blood was sampled from a radial artery and from the portal and right renal vein. Amino acid concentrations and enrichments were measured, and net fluxes of [(15)N]-labeled substrates across the portal drained viscera (PDV) and kidneys were calculated from arteriovenous differences and plasma flow. RESULTS: Arterial [(15)N]glutamine enrichments were significantly lower during enteral tracer infusion (tracer-to-tracee ratio [labeled vs unlabeled substrate, TTR%] IV: 6.66 +/- 0.35 vs EN: 3.04 +/- 0.45; p < .01), reflecting first-pass intestinal metabolism of glutamine during absorption. Compared with IV administration, enteral administration of the glutamine tracer resulted in a significantly higher intestinal fractional extraction of [(15)N]glutamine (IV: 0.15 +/- 0.03 vs EN: 0.44 +/- 0.08 mumol/kg/h; p < .01). Furthermore, enteral administration of the glutamine tracer resulted in higher arterial enrichments of [(15)N]citrulline (TTR% IV: 5.52 +/- 0.44 vs EN: 8.81 +/- 1.1; p = .02), and both routes of administration generated a significant enrichment of [(15)N]arginine (TTR% IV: 1.43 +/- 0.12 vs EN: 1.68 +/- 0.18). This was accompanied by intestinal release of [(15)N]citrulline across the PDV, which was higher with enteral glutamine (IV: 0.38 +/- 0.07 vs EN: 0.72 +/- 0.11 mumol/kg/h; p = .02), and subsequent [(15)N]arginine release in both groups. CONCLUSIONS: In humans, the gut preferably takes up enterally administered glutamine compared with intravenously provided glutamine. The route of administration, enteral or IV, affects the quantitative conversion of glutamine into citrulline and subsequent renal arginine synthesis in humans.