BACKGROUND: We previously confirmed in humans the existence of a pathway of glutamine into citrulline and arginine, which is preferentially stimulated by luminally provided glutamine. However, because glutamine is unstable, we tested this pathway with a stable dipeptide of glutamine. OBJECTIVES: The objectives were to explore whether alanyl-glutamine contributes to the synthesis of arginine in humans and whether this depends on the route of administration. DESIGN: The study was conducted under postabsorptive conditions during surgery. Sixteen patients received alanyl-[2-(15)N]glutamine enterally or intravenously together with intravenously administered stable-isotope tracers of citrulline and arginine. Blood was collected from an artery, the portal vein, a hepatic vein, and the right renal vein. Arterial and venous enrichments and (tracer) net balances of alanyl-glutamine and glutamine, citrulline, and arginine across the portal drained viscera, liver, and kidneys were determined. Parametric tests were used to test results (mean +/- SEM). P < 0.05 was considered significant. RESULTS: Twice as much exogenous glutamine was used for the synthesis of citrulline when alanyl-glutamine was provided enterally (5.9 +/- 0.6%) than when provided intravenously (2.8 +/- 0.3%) (P < 0.01). Consequently, twice as much exogenous glutamine was used for the synthesis of arginine when alanyl-glutamine was provided enterally (5 +/- 0.7%) than when provided intravenously (2.4 +/- 0.2%) (P < 0.01). However, results at the organ level did not explain the differences due to route of administration. CONCLUSIONS: Alanyl-glutamine contributes to the de novo synthesis of arginine, especially when provided enterally. A stable-isotope study using a therapeutic dose of alanyl-glutamine is needed to investigate the clinical implications of this finding.