The impaired ability of the autonomic nervous system to respond to hypoglycemia is termed "hypoglycemia-associated autonomic failure" (HAAF). This life-threatening phenomenon results from at least two recent episodes of hypoglycemia, but the pathology underpinning HAAF remains largely unknown. Although naloxone appears to improve hypoglycemia counterregulation under controlled conditions, hypoglycemia prevention remains the current mainstay therapy for HAAF. Epinephrine-synthesizing neurons in the rostroventrolateral (C1) and dorsomedial (C3) medulla project to the subset of sympathetic preganglionic neurons that regulate peripheral epinephrine release. Here we determined whether or not C1 and C3 neuronal activation is impaired in HAAF and whether or not 1 wk of hypoglycemia prevention or treatment with naloxone could restore C1 and C3 neuronal activation and improve HAAF. Twenty male Sprague-Dawley rats (250-300 g) were used. Plasma epinephrine levels were significantly increased after a single episode of hypoglycemia ( = 4; 5,438 ± 783 pg/ml vs. control 193 ± 27 pg/ml, < 0.05). Repeated hypoglycemia significantly reduced the plasma epinephrine response to subsequent hypoglycemia ( = 4; 2,179 ± 220 pg/ml vs. 5,438 ± 783 pg/ml, < 0.05). Activation of medullary C1 ( = 4; 50 ± 5% vs. control 3 ± 1%, < 0.05) and C3 ( = 4; 45 ± 5% vs. control 4 ± 1%, < 0.05) neurons was significantly increased after a single episode of hypoglycemia. Activation of C1 ( = 4; 12 ± 3%, < 0.05) and C3 ( = 4; 19 ± 5%, < 0.05) neurons was significantly reduced in the HAAF groups. Hypoglycemia prevention or treatment with naloxone did not restore the plasma epinephrine response or C1 and C3 neuronal activation. Thus repeated hypoglycemia reduced the activation of C1 and C3 neurons mediating adrenal medullary responses to subsequent bouts of hypoglycemia.