Technical Abstract: Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton, versus nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, 2-15N and 5-15N glutamine and 15N ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled almost equally the three nitrogen groups of citrulline. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta nitrogen. D5 glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was poorly utilized accounting only for 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25 fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-13Cn tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for ~40% of the circulating citrulline. Proline contribution was minor (3.4%) and glutamine was negligible (0.4%). The glutamine tracer, however, resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.