The contribution of net hepatic glycogenolysis to overall glucose production during a physiological increment in the plasma glucagon concentration was measured in six healthy subjects (18–24 years, 68–105 kg) after an overnight fast. Glucagon (∼3 ng · kg⁻¹ · min⁻¹), somatostatin (0.1 μg · kg⁻¹ · min⁻¹), and insulin (0.9 pmol · kg⁻¹ · min⁻¹) were infused for 3 h. Liver glycogen concentration was measured at 15-min intervals during this period using ¹³C-labeled nuclear magnetic resonance spectroscopy, and liver volume was assessed from magnetic resonance images. The rate of net hepatic glycogenolysis was calculated from the decrease in liver glycogen concentration over time, multiplied by the liver volume. The rate of glucose appearance (R_a) was calculated from [3-³H]glucose turnover data using a two-compartment model of glucose kinetics. Plasma glucagon concentration rose from 136 ± 18 to 304 ± 57 ng/1 and plasma glucose concentration rose from 5.6 ± 0.1 to 10.4 ± 0.9 mmol/1 on initiation of the infusions. Mean baseline R_a was 11.8 ± 0.4 μmol · kg⁻¹ · min⁻¹, increased rapidly after the beginning of the infusions, reaching its highest value after 20–40 min, and returned to baseline by 140 min. Liver glycogen concentration decreased almost linearly (from 300 ± 19 mmol/1 liver at baseline to 192 ± 20 mmol/1 liver at t = 124 min) during 2 h after the beginning of the infusions, and the calculated mean rate of net hepatic glycogenolysis was 21.7 ± 3.6 μmol · kg⁻¹ · min⁻¹. Mean R_a during the same time period was 22.8 ± 2.3 μmol · kg⁻¹ · min⁻¹. Thus, net hepatic glycogenolysis accounted for 93 ± 9% of R_a. In conclusion, during the initial response to a physiological increment in plasma glucagon, 1) net hepatic glycogenolysis accounts for virtually all of the increase in hepatic glucose production, and 2) glucagon's evanescent effect on hepatic glucose production is not caused by depletion of hepatic glycogen stores.