The roles of glycogenolysis and gluconeogenesis in sustaining glucose production during insulin-induced hypoglycemia were assessed in overnight-fasted conscious dogs. Insulin was infused intraportally for 3 h at 5 mU.kg-1.min-1 in five animals, and glycogenolysis and gluconeogenesis were measured by using a combination of tracer [( 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous difference techniques. In response to the elevated insulin level (263 +/- 39 microU/ml), plasma glucose level fell (41 +/- 3 mg/dl), and levels of the counterregulatory hormones glucagon, epinephrine, norepinephrine, and cortisol increased (91 +/- 29 to 271 +/- 55 pg/ml, 83 +/- 26 to 2356 +/- 632 pg/ml, 128 +/- 31 to 596 +/- 81 pg/ml, and 1.5 +/- 0.4 to 11.1 +/- 1.0 micrograms/dl, respectively; for all, P less than .05). Glucose production fell initially and then doubled (3.1 +/- 0.3 to 6.1 +/- 0.5 mg.kg-1.min-1; P less than .05) by 60 min. Net hepatic gluconeogenic precursor uptake increased approximately eightfold by the end of the hypoglycemic period. By the same time, the efficiency with which the liver converted the gluconeogenic precursors to glucose rose twofold. Five control experiments in which euglycemia was maintained by glucose infusion during insulin administration (5.0 mU.kg-1.min-1) provided baseline data. Glycogenolysis accounted for 69-88% of glucose production during the 1st h of hypoglycemia, whereas gluconeogenesis accounted for 48-88% of glucose production during the 3rd h of hypoglycemia. These data suggest that gluconeogenesis is the key process for the normal counterregulatory response to prolonged and marked hypoglycemia.