L-Proline and L-glutamine were used to probe the inverse relationship between glycogenesis and ureagenesis in isolated, perfused livers from 48-h fasted rats. Both amino acids may provide nitrogen in the form of NH+4 for carbamyl-P synthesis. However, one molecule of glutamine may provide additionally for the synthesis of one molecule of the urea cycle substrate L-aspartate, but proline can provide for the synthesis of a molecule of NH+4 or one molecule of aspartate on an either/or basis only. In all perfusates, glucose was initially 30 mM (to favor phosphotransferase activity of glucose-6-phosphatase) and 0.5 mM 3-mercaptopicolinate was present (to inhibit glyconeogenesis from endogenous substrates, from the added amino acids, and via the indirect pathway). Glycogenesis from glucose, perfusate and hepatic urea formation, and levels of hepatic glucose-6-P, citrulline, PPi, and carbamyl-P were measured. The addition of glutamine to the perfusate markedly stimulated the urea cycle, but not glycogenesis. Hepatic urea level, perfusate urea concentration, and hepatic citrulline and PPi increased while carbamyl-P content decreased. In contrast, proline stimulated glycogenesis from glucose, but not ureagenesis. In the proline-supplemented compared with glutamine group, hepatic glycogenesis and carbamyl-P content increased; hepatic glucose-6-P levels showed a tendency toward increase; and hepatic urea formation, hepatic citrulline, and PPi levels were decreased. These observations are interpreted to support an hepatic mechanism whereby the relative availability of carbamyl-P to the urea cycle and as a substrate for glucose phosphorylation via phosphotransferase activity of the glucose-6-phosphatase system preliminary to glycogenesis from glucose is a major metabolic determinant.