The rate of net hepatic glycogenolysis was assessed in humans by serially measuring hepatic glycogen concentration at 3- to 12-hour intervals during a 68-hour fast with ¹³C nuclear magnetic resonance spectroscopy. The net rate of gluconeogenesis was calculated by subtracting the rate of net hepatic glycogenolysis from the rate of glucose production in the whole body measured with tritiated glucose. Gluconeogenesis accounted for 64 ± 5% (mean ± standard error of the mean) of total glucose production during the first 22 hours of fasting. In the subsequent 14-hour and 18-hour periods of the fast, gluconeogenesis accounted for 82 ± 5% and 96 ± 1% of total glucose production, respectively. These data show that gluconeogenesis accounts for a substantial fraction of total glucose production even during the first 22 hours of a fast in humans.