Antecedent insulin-induced hypoglycemia (IIH) reduces adrenomedullary responses (AMR) to subsequent bouts of hypoglycemia. The ventromedial hypothalamus (VMH: arcuate [ARC] + ventromedial nuclei [VMN]) contains glucosensing neurons which are thought to be mediators of this AMR. Since type 1 diabetes mellitus often begins in childhood, we used juvenile (4-5 wk old) rats and demonstrated that a single bout of IIH (5 U/kg, s.c.) reduced plasma glucose level by 24% and peak epinephrine by 59% 1 d later. This dampened AMR was associated with 46% higher mRNA for VMH glucokinase (GK), a key mediator of neuronal glucosensing. Compared to neurons from saline-injected rats, VMN glucose excited (GE) neurons from insulin-injected rats demonstrated a left-shift in their glucose responsiveness (EC50 saline- 0.45; insulin- 0.10 mmol/l; P=0.05) and a 31% higher maximal activation by glucose (P=0.05), although this maximum occurred at a higher glucose concentration (saline- 0.7; insulin- 1.5 mmol/l). While EC50 values did not differ, ARC GE neurons had 19% higher maximal activation which occurred at a lower glucose concentration (saline- 2.5; insulin- 1.5 mmol/l) than those from saline-injected rats. In addition, ARC glucose inhibited neurons from insulin-injected rats were maximally inhibited at a five-fold lower glucose concentration (saline- 2.5; insulin- 0.5 mmol/l), although this inhibition declined above 0.5 mmol/l glucose. These data suggest that the increased VMH GK seen following antecedent IIH may contribute to the increased responsiveness of VMH glucosensing neurons to glucose and contribute to the associated blunting of the AMR.