Recurrent hypoglycemia impairs hormonal counterregulatory responses (CRRs) to further bouts of hypoglycemia. The hypothalamus and hindbrain are both critical for sensing hypoglycemia and triggering CRRs. Hypothalamic glucose sensing sites are implicated in the pathogenesis of defective CRRs; however, the contribution of hindbrain glucose sensing has not been elucidated. Using a rat model, we compared the effect of antecedent glucoprivation targeting hindbrain or hypothalamic glucose sensing sites with the effect of antecedent recurrent hypoglycemia on CRR to hypoglycemia induced 24 h later. Recurrent hypoglycemia decreased sympathoadrenal (1,470 +/- 325 vs. 3,811 +/- 540 pg/ml in controls [t = 60 min], P = 0.001) and glucagon secretion (222 +/- 43 vs. 494 +/- 56 pg/ml in controls [t = 60]), P = 0.003) in response to hypoglycemia. Antecedent 5-thio-glucose (5TG) injected into the hindbrain did not impair sympathoadrenal (3,806 +/- 344 pg/ml [t = 60]) or glucagon (513 +/- 56 pg/ml [t = 60]) responses to subsequent hypoglycemia. However, antecedent 5TG delivered into the third ventricle was sufficient to blunt CRRs to hypoglycemia. These results show that hindbrain glucose sensing is not involved in the development of defective CRRs. However, neural substrates surrounding the third ventricle are particularly sensitive to glucoprivic stimulation and may contribute importantly to the development of defective CRRs.