Antecedent hypoglycemic episodes reduce the counterregulatory neuroendocrine response to hypoglycemia. The role of insulin in the mechanism responsible for the antecedent hypoglycemia causing subsequent counterregulatory failure has not been elucidated. We performed antecedent hypoglycemic clamps (56 mg/dL) lasting 2 h with differing degrees of hyperinsulinemia, which were followed by 6-h stepwise hypoglycemic clamps (76-66-56-46 mg/dL) on the next day. Experiments were carried out in 30 young, healthy men. Fifteen of these subjects were tested on 2 occasions. On 1 occasion the antecedent hypoglycemia was induced by insulin infusion at a rate of 1.5 mU/min x kg (low insulin-ante-hypo); on the other occasion the insulin infusion rate was 15.0 mU/min x kg (high insulin-ante-hypo). Both sessions were separated by at least 4 weeks, and their order was balanced across subjects. The remaining 15 subjects (control group) received the same stepwise hypoglycemic clamp as the other subjects, but without antecedent hypoglycemia. During the stepwise hypoglycemic clamp, the counterregulatory increases in ACTH, cortisol, and norepinephrine were significantly blunted after the low insulin-ante-hypo (P < 0.01, P < 0.05, and P < 0.05, respectively) but not after the high insulin-ante-hypo (P = 0.12, P = 0.92, and P = 0.19, respectively) compared to that in the control group. The cortisol, norepinephrine, and glucagon responses were greater after the high than after the low insulin-ante-hypo (all P < 0.05). In conclusion, the present study clearly demonstrates that even a single episode of mild hypoglycemia reduces neuroendocrine counterregulation 18-24 h later. Insulin has a moderate protective effect on subsequent counterregulation.