We previously showed that agmatine stimulated hepatic ureagenesis. In this study, we sought to determine whether the action of agmatine is mediated via cAMP signaling. A pilot experiment demonstrated that the phosphodiesterase inhibitor, 3-isobutylmethylxanthine (IBMX), inhibited urea synthesis albeit increased [cAMP]. Thus, we hypothesized that IBMX inhibits hepatic urea synthesis independent of [cAMP]. We further theorized that agmatine would negate the IBMX action and improve ureagenesis. Experiments were carried out with isolated mitochondria and (15)NH(4)Cl to trace [(15)N]citrulline production or [5-(15)N]glutamine and a rat liver perfusion system to trace ureagenesis. The results demonstrate that IBMX induced the following: (i) inhibition of the mitochondrial respiratory chain and diminished O(2) consumption during liver perfusion; (ii) depletion of the phosphorylation potential and overall hepatic energetic capacity; (iii) inhibition of [(15)N]citrulline synthesis; and (iv) inhibition of urea output in liver perfusion with little effect on [N-acetylglutamate]. The results indicate that IBMX directly and specifically inhibited complex I of the respiratory chain and carbamoyl-phosphate synthase-I (CPS-I), with an EC(50) about 0.6 mm despite a significant elevation of hepatic [cAMP]. Perfusion of agmatine with IBMX stimulated O(2) consumption, restored hepatic phosphorylation potential, and significantly stimulated ureagenesis. The action of agmatine may signify a cascade effect initiated by increased oxidative phosphorylation and greater ATP synthesis. In addition, agmatine may prevent IBMX from binding to one or more active site(s) of CPS-I and thus protect against inhibition of CPS-I. Together, the data may suggest a new experimental application of IBMX in studies of CPS-I malfunction and the use of agmatine as intervention therapy.