Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of L-[guanidino-¹⁵N₂]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with ¹⁵N-labeled glutamine and unlabeled ammonia or ¹⁵NH₄Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-¹⁵N₂]agmatine is formed in the liver from perfusate L-[guanidino-¹⁵N₂]arginine (~90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of ¹⁵N-labeled NAG and [¹⁵N]urea from ¹⁵N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of ¹⁵N-labeled NAG and [¹⁵N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.