To assess the role of counterregulatory hormones per se in the response to continuous insulin infusion, overnight-fasted dogs were given 5 mU.kg-1.min-1 insulin intraportally either alone (INS, n = 5), with glucose to maintain euglycemia (INS + GLU, n = 5), or with glucose and hormone replacement [i.e., glucagon, epinephrine, norepinephrine, and cortisol infusions (INS + GLU + HR, n = 6)]. The increases in counterregulatory hormones that occurred during insulin-induced hypoglycemia were simulated in the latter group. In this way, it was possible to separate the effects of hypoglycemia per se from those due to the associated counterregulatory hormone response. Glycogenolysis and gluconeogenesis were measured with a combination of tracer ([ 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous (AV) difference techniques during a 40-min control and a 180-min experimental period. Insulin levels increased similarly in all groups (to congruent to 250 microU/ml), whereas plasma glucose levels decreased in INS (115 +/- 3 to 41 +/- 3 mg/dl; P less than .05) and rose slightly in both INS + GLU (108 +/- 2 to 115 +/- 4 mg/dl; P less than .05) and INS + GLU + HR (111 +/- 3 to 120 +/- 3 mg/dl; P less than .05) due to glucose infusion. Glucagon, epinephrine, norepinephrine, and cortisol were replaced in INS + GLU + HR so that the increments in their levels were 102 +/- 6, 106 +/- 14, 117 +/- 9, and 124 +/- 37%, respectively, of their increments in INS. At no time was there a significant difference between the hormone levels in INS and INS + GLU + HR. The rise in the counterregulatory hormones per se accounted for only half (53 +/- 9% by the AV difference method and 54 +/- 10% by tracer method) of the glucose production associated with hypoglycemia resulting from insulin infusion. The rate and efficiency of alanine conversion to glucose in the hormone-replacement studies were only 29 +/- 10 and 50 +/- 27% of what occurred during hypoglycemia induced by insulin infusion. In conclusion, the counterregulatory hormones alone (i.e., without accompanying hypoglycemia) can account for only 50% of the glucose production that is present during insulin-induced hypoglycemia. The remaining 50%, therefore, must result from effects of hypoglycemia other than its ability to trigger hormone release.