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 β-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 μg/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 β-cell) impairs defense mechanisms against glucose deprivation and therefore could contribute to defects in glucose counterregulation.