To assess the contribution of the human kidney to gluconeogenesis (GN) and its role in conversion of glutamine and alanine to glucose, we used a combination of isotopic and organ balance techniques in nine normal postabsorptive volunteers and measured both overall and renal incorporation of these precursors into glucose before and after infusion of epinephrine. In the postabsorptive basal state, renal incorporation of glutamine (27±2 μmol/min) and alanine (2.1±0.5 μmol/min) into glucose accounted for 72.8±3.3 and 3.9±0.5% of their overall incorporation into glucose (37±2 and 51±6 μmol/min, respectively) and 19.0±3.5 and 1.4±0.2%, respectively, of overall renal glucose release. Infusion of epinephrine, which increased systemic and renal glucose release more than twofold (P< 0.001), increased overall glutamine and alanine incorporation into glucose (both P< 0.001) and increased renal GN from glutamine (P< 0.001) but not from alanine (P= 0.15). Renal glutamine GN now accounted for 90.3±4.0% of overall glutamine GN (P= 0.01 vs. basal), whereas renal alanine GN still accounted for only 4.8±1.7% of overall alanine GN (P= 0.36 vs. basal). With the assumption that kidney and liver are the only gluconeogenic organs in humans, these results indicate that glutamine GN occurs primarily in kidney, whereas alanine GN occurs almost exclusively in liver. Isotopic studies of glutamine and alanine incorporation into plasma glucose may provide a selective, noninvasive method to assess hepatic and renal GN.