Relative contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production during the first 12 h of a fast were studied in 13 healthy volunteers by noninvasively measuring hepatic glycogen content using 13C nuclear magnetic resonance spectroscopy. Rates of net hepatic glycogenolysis were calculated by multiplying the change in liver glycogen content with liver volume determined by magnetic resonance imaging. Rates of gluconeogenesis were calculated as the difference between rates of glucose production determined with an infusion of [6,6-2H]-glucose and net hepatic glycogenolysis. At 6 P.M. a liquid mixed meal (1,000 kcal; 60% as glucose) was given, to which [2-2H]glucose was added to trace glucose absorption. Hepatic glycogen content was measured between 11 P.M. and 1 A.M. and between 3 and 6 A.M. At 11 P.M. the concentration was 470 mM and it decreased linearly during the night. The mean liver volume was 1.47 +/- 0.06 liters. Net hepatic glycogenolysis (5.8 +/- 0.8 mumol.kg body wt-1.min-1) accounted for, on average, 45 +/- 6% and gluconeogenesis for 55 +/- 6% of the rate of whole body glucose production (12.6 +/- 0.6 mumol.kg body wt-1.min-1). In conclusion, this study shows that, even early in the phase of the postabsorptive period when liver glycogen stores are maximal, gluconeogenesis contributes approximately 50% to hepatic glucose production.