There is considerable controversy recently in identifying dietary precursors for arginine synthesis. We have previously shown in human neonates and piglets that proline is the sole dietary precursor for arginine synthesis. It is unclear in adult humans whether proline is a dietary precursor for arginine. We performed a multi-tracer stable isotope study in adults using (15)N(2)-ureido arginine and (15)N proline to elucidate synthesis of citrulline and arginine and determine whether proline is a precursor for arginine. Primed, intermittent infusions of the labeled amino acids were given enterally to 5 healthy men consuming a standardized milkshake diet. Blood was sampled during plateau enrichment between 1.5 and 3 h. Plasma enrichment occurred for both tracers, giving enteral turnover estimates of 93 μmol · kg(-1) · h(-1) for arginine and 154 μmol · kg(-1) · h(-1) for proline. Appearance of the label from proline in arginine and the intermediaries, ornithine and citrulline, was measured in all participants. The rate of synthesis of arginine from proline was 3.7 μmol · kg(-1) · h(-1), which is estimated to be ~40% of newly synthesized arginine. In this first study in adult humans using an enteral proline tracer, we have demonstrated synthesis of arginine from this dietary amino acid. Therefore, as in newborns, proline must now be considered a dietary precursor for arginine in healthy adults.