The ventromedial hypothalamic nucleus (VMH) is necessary for the integrated hormonal response to hypoglycemia. To determine the role of the VMH as a glucose sensor, we performed experiments designed to specifically prevent glucopenia in the VMH, while producing hypoglycemia elsewhere. We used awake chronically catheterized rats, in which local VMH glucose perfusion (100 mM or 15 mM of D-glucose) was combined with a sequential euglycemic-hypoglycemic clamp. In two control groups the VMH was perfused either with (a) an iso-osmotic solution lacking glucose, or with (b) nonmetabolizable L-glucose (100 mM). During systemic hypoglycemia glucagon and catecholamine concentrations promptly increased in the control animals perfused with either 100 mM L-glucose or the iso-osmotic solution lacking glucose. In contrast, glucagon, epinephrine and norepinephrine release was inhibited in the animals in which the VMH was perfused with D-glucose; hormonal secretion was partially suppressed by the VMH perfusion with 15 mM D-glucose and suppressed by approximately 85% when the VMH was perfused with 100 mM D-glucose, as compared with the control groups. We conclude that the VMH must sense hypoglycemia for full activation of catecholamine and glucagon secretion and that it is a key glucose sensor for hypoglycemic counterregulation.