It has been postulated that specialized glucose-sensing neurons in the ventromedial hypothalamus (VMH) are able to detect falling blood glucose and trigger the release of counterregulatory hormones during hypoglycemia. The molecular mechanisms used by glucose-sensing neurons are uncertain but may involve cell surface ATP-sensitive K(+) channels (K(ATP) channels) analogous to those of the pancreatic beta-cell. We examined whether the delivery of sulfonylureas directly into the brain to close K(ATP) channels would modulate counterregulatory hormone responses to either brain glucopenia (using intracerebroventricular 5-thioglucose) or systemic hypoglycemia in awake chronically catheterized rats. The closure of brain K(ATP) channels by global intracerebroventricular perfusion of sulfonylurea (120 ng/min glibenclamide or 2.7 microg/min tolbutamide) suppressed counterregulatory (epinephrine and glucagon) responses to brain glucopenia and/or systemic hypoglycemia (2.8 mmol/l glucose clamp). Local VMH microinjection of a small dose of glibenclamide (0.1% of the intracerebroventricular dose) also suppressed hormonal responses to systemic hypoglycemia. We conclude that hypothalamic K(ATP) channel activity plays an important role in modulating the hormonal counterregulatory responses triggered by decreases in blood glucose. Our data suggest that closing of K(ATP) channels in the VMH (much like the beta-cell) impairs defense mechanisms against glucose deprivation and therefore could contribute to defects in glucose counterregulation.