The mechanisms involved in the release of glucagon in response to hypoglycemia are unclear. Proposed mechanisms include the activation of the autonomic nervous system via glucose-sensing neurons in the central nervous system, via the regulation of glucagon secretion by intra-islet insulin and zinc concentrations, or via direct ionic control, all mechanisms that involve high-affinity sulfonylurea receptor/inwardly rectifying potassium channel-type ATP-sensitive K(+) channels. Patients with congenital hyperinsulinism provide a unique physiological model to understand glucagon regulation. In this study, we compare serum glucagon responses to hyperinsulinemic hypoglycemia versus nonhyperinsulinemic hypoglycemia. In the patient group (n = 20), the mean serum glucagon value during hyperinsulinemic hypoglycemia was 17.6 +/- 5.7 ng/l compared with 59.4 +/- 7.8 ng/l in the control group (n = 15) with nonhyperinsulinemic hypoglycemia (P < 0.01). There was no difference between the serum glucagon responses in children with diffuse, focal, and diazoxide-responsive forms of hyperinsulinism. The mean serum epinephrine and norepinephrine concentrations in the hyperinsulinemic group were 2,779 +/- 431 pmol/l and 2.9 +/- 0.7 nmol/l and appropriately rose despite the blunted glucagon response. In conclusion, the loss of ATP-sensitive K(+) channels and or elevated intraislet insulin cannot explain the blunted glucagon release in all patients with congenital hyperinsulinism. Other possible mechanisms such as the suppressive effect of prolonged hyperinsulinemia on alpha-cell secretion should be considered.