To determine the mechanism(s) by which insulin inhibits endogenous glucose production (EGP) in nondiabetic humans, insulin was infused at rates of 0.25, 0.375, or 0.5 mU. kg(-1). min(-1) and glucose was clamped at approximately 5.5 mmol/l. EGP, gluconeogenesis, and uridine-diphosphoglucose (UDP)-glucose flux were measured using [3-(3)H]glucose, deuterated water, and the acetaminophen glucuronide methods, respectively. An increase in insulin from approximately 75 to approximately 100 to approximately 150 pmol/l ( approximately 12.5 to approximately 17 to approximately 25 microU/ml) resulted in progressive (ANOVA; P < 0.02) suppression of EGP (13.1 +/- 1.3 vs. 11.7 +/- 1.03 vs. 6.4 +/- 2.15 micromol x kg(-1) x min(-1)) that was entirely due to a progressive decrease (ANOVA; P < 0.05) in the contribution of glycogenolysis to EGP (4.7 +/- 1.7 vs. 3.4 +/- 1.2 vs. -2.1 +/- 1.3 micro mol x kg(-1) x min(-1)). In contrast, both the contribution of gluconeogenesis to EGP (8.4 +/- 1.0 vs. 8.3 +/- 1.1 vs. 8.5 +/- 1.3 micro mol x kg(-1) x min(-1)) and UDP-glucose flux (5.0 +/- 0.4 vs. 5.0 +/- 0.3 vs. 4.0 +/- 0.5 micro mol x kg(-1) x min(-1)) remained unchanged. The contribution of the direct (extracellular) pathway to UDP-glucose flux was minimal and constant during all insulin infusions. We conclude that higher insulin concentrations are required to suppress the contribution of gluconeogenesis of EGP than are required to suppress the contribution of glycogenolysis to EGP in healthy nondiabetic humans. Since suppression of glycogenolysis occurred without a decrease in UDP-glucose flux, this implies that insulin inhibits EGP, at least in part, by directing glucose-6-phosphate into glycogen rather than through the glucose-6-phosphatase pathway.