To further characterize mechanisms of glucose counterregulation in man, the effects of pharmacologically induced deficiencies of glucagon, growth hormone, and catecholamines (alone and in combination) on recovery of plasma glucose from insulin-induced hypoglycemia and attendant changes in isotopically ([3-3H]glucose) determined glucose fluxes were studied in 13 normal subjects. In control studies, recovery of plasma glucose from hypoglycemia was primarily due to a compensatory increase in glucose production; the temporal relationship of glucagon, epinephrine, cortisol, and growth hormone responses with the compensatory increase in glucose appearance was compatible with potential participation of all these hormones in acute glucose counterregulation. Infusion of somatostatin (combined deficiency of glucagon and growth hormone) accentuated insulin-induced hypoglycemia (plasma glucose nadir: 36 ± 2 ng/dl during infusion of somatostatin vs. 47 ± 2 mg/dl in control studies, P<0.01) and impaired restoration of normoglycemia (plasma glucose at min 90: 73 ± 3 mg/dl at end of somatostatin infusion vs. 92 ± 3 mg/dl in control studies, P<0.01). This impaired recovery of plasma glucose was due to blunting of the compensatory increase in glucose appearance since glucose disappearance was not augmented, and was attributable to suppression of glucagon secretion rather than growth hormone secretion since these effects of somatostatin were not observed during simultaneous infusion of somatostatin and glucagon whereas infusion of growth hormone along with somatostatin did not prevent the effects of somatostatin. The attenuated recovery of plasma glucose from hypoglycemia observed during somatostatin-induced glucagon deficiency was associated with plasma epinephrine levels twice those observed in control studies. Infusion of phentolamine plus propranolol (combined α- and β-adrenergic blockade) had no effect on plasma glucose or glucose fluxes after insulin administration. However, infusion of somatostatin along with both phentolamine and propranolol further impaired recovery of plasma glucose from hypoglycemia compared to that observed with somatostatin alone (plasma glucose at end of infusions: 52 ± 6 mg/dl for somatostatin-phentolamine-propranolol vs. 72±5 mg/dl for somatostatin alone, P<0.01); this was due to further suppression of the compensatory increase in glucose appearance (maximal values: 1.93 ± 0.41 mg/kg per min for somatostatin-phentolamine-propranolol vs. 2.86 ± 0.32 mg/kg per min for somatostatin alone, P<0.05). These results indicate that in man restoration of normoglycemia after insulin-induced hyperglycemia is primarily due to a compensatory increase in glucose production; intact glucagon secretion, but not growth hormone secretion, is necessary for normal glucose counterregulation, and adrenergic mechanisms do not normally play an essential role in this process but become critical to recovery from hypoglycemia when glucagon secretion is impaired.