Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by ¹H,²H, and ¹³C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-¹³C₂]glucose,²H₂O, and [U-¹³C₃]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-¹³C₂] glucose by ¹H and ¹³C NMR indicated that GP in 16-h-fasted humans was 10.7 ± 0.9 μmol · kg⁻¹ · min⁻¹.²H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative ²H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 ± 0.7, 4.8 ± 1.0, and 0.4 ± 0.3 μmol · kg⁻¹ · min⁻¹, respectively). Interestingly, ¹³C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 ± 9.8 vs. 37.5 ± 7.9 μmol · kg⁻¹ · min⁻¹), PEP recycling flux (59.1 ± 9.8 vs. 27.8 ± 6.8 μmol · kg⁻¹ · min⁻¹), and TCA cycle flux (10.9 ± 1.4 vs. 5.4 ± 1.4 μmol · kg⁻¹ · min⁻¹). These differences may reflect zonation of propionate metabolism in the liver.