Dietary arginine is the main dietary precursor for citrulline synthesis, but it is not known if other precursors can compensate when arginine is absent in the diet. To address this question, the contributions of plasma and dietary precursors were determined by using multitracer protocols in conscious mice infused i.g. either an arginine-sufficient diet [Arg(+)] or an arginine-free diet [Arg(-)]. The plasma entry rate of citrulline and arginine did not differ between the 2 diet groups (156 +/- 6 and 564 +/- 30 micromol kg(-1) h(-1), respectively); however, the entry rate of ornithine was greater in the mice fed the Arg(+) than the Arg(-) diet (332 +/- 33 vs. 180 +/- 16 micromol kg(-1) h(-1)). There was a greater utilization of plasma ornithine for the synthesis of citrulline (49 +/- 4 vs. 36 +/- 3 micromol kg(-1) h(-1), 30 +/- 3% vs. 24 +/- 2% of citrulline entry rate) in the mice fed the Arg(-) diet than the Arg(+) diet. The utilization of plasma arginine did not differ between the 2 diet groups for citrulline synthesis, either through plasma ornithine (approximately 29 +/- 3 micromol kg(-1) h(-1)) or at the site of citrulline synthesis (approximately 12 +/- 3 micromol kg(-1) h(-1)). The contribution of dietary proline to the synthesis of citrulline was mainly at the site of citrulline production (17 +/- 1 micromol kg(-1) h(-1)), rather than through plasma ornithine (5 +/- 0.4 micromol kg(-1) h(-1)). Dietary glutamine was utilized only at the site of citrulline synthesis (4 +/- 0.2 micromol kg(-1) h(-1)). Dietary glutamine and proline made a greater contribution to the synthesis of citrulline in mice fed the Arg(-) diet but remained minor sources for citrulline production. Plasma arginine and ornithine are able to support citrulline synthesis during arginine-free feeding.