Technical Abstract: Recent isotopic tracer studies in mice, piglets, and humans have produced conflicting results as to the main carbon skeleton precursor for citrulline and arginine synthesis. This may be due in part to the different tracers infused and models utilized to interpret the stable isotope data. Furthermore, previous studies usually investigated a single precursor, which prevented the direct comparison among multiple precursors. To further elucidate the contribution of different precursors to citrulline synthesis, all possible enteral and plasma precursors of citrulline were studied in a mouse model during the postabsorptive and postprandrial period utilizing multitracer protocols. In addition, three different models were utilized in order to interpret the stable isotope data. The utilization of the classic precursor-product equation, developed for intravenously infused tracers but used to include also intragastrically tracers, grossly overestimated the contribution of enteral precursors. Regardless of the model employed, dietary and plasma arginine were the main precursors for citrulline synthesis during feeding and plasma arginine during fasting. The contribution of arginine was directly at the site of citrulline synthesis and through plasma ornithine. The predominant role of arginine and ornithine seen in this study supports the observations in mice, piglets, and humans, suggesting that ornithine amino transferase is a pivotal enzyme in this pathway.