Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single (2)H-NMR spectrum is demonstrated with 6-hr and 24-hr fasted rats. Animals were administered [1-(2)H, 1-(13)C]glucose, a novel tracer of glucose turnover, and (2)H(2)O. Plasma glucose enrichment from both tracers was quantified by (2)H-NMR analysis of monoacetone glucose. The 6-hr fasted group (n = 7) had EGP rates of 95.6 +/- 13.3 micromol/kg/min, where 56.2 +/- 7.9 micromol/kg/min were derived from PEP; 12.1 +/- 2.1 micromol/kg/min from glycerol, and 32.1 +/- 4.9 micromol/kg/min from glycogen. The 24-hr fasted group (n = 7) had significantly lower EGP rates (52.8 +/- 7.2 micromol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 +/- 5.9 micromol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 +/- 3.9 and 8.5 +/- 1.2 micromol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from (2)H(2)O and (13)C-glucose tracers.