Hypoglycemia-associated autonomic failure (HAAF) occurs commonly in patients with longstanding diabetes, placing affected patients at increased risk for severe hypoglycemia. Previous studies have suggested that hypoglycemia-induced hypercortisolemia may be responsible for blunting subsequent sympathoadrenal responses to hypoglycemia; however, this view remains highly controversial. In this work, we sought to better define the role of antecedent hypercortisolemia in generating HAAF, using two complimentary experimental models in nondiabetic human subjects: 1) antecedent hydrocortisone infusions (simulating physiologic cortisol responses to hypoglycemia) and 2) antecedent hypoglycemia, with and without concurrent blockade of endogenous cortisol production using oral metyrapone. Our results showed no effect of antecedent hypercortisolemia on epinephrine responses to subsequent hypoglycemia (area under the curve/time 280 +/- 53 vs. 337 +/- 57 pg/ml, P = 0.16). Of particular importance, selective blockade of endogenous cortisol production during antecedent hypoglycemia had no effect on subsequent counterregulatory responses to hypoglycemia. Compared with epinephrine responses following antecedent euglycemia (area under the curve/time 312 +/- 38 pg/ml), epinephrine responses were comparably blunted following antecedent hypoglycemia, regardless of whether concurrent metyrapone blockade was employed (198 +/- 28 vs. 192 +/- 28 pg/ml, P = NS). Similar results were obtained for glucagon and ACTH levels. Considered together, these observations provide strong evidence that hypoglycemia-induced hypercortisolemia is not primarily responsible for the generation of HAAF.