The impaired epinephrine and glucagon responses to hypoglycemia often found in patients with insulindependent diabetes mellitus (IDDM) may be due to autonomic neuropathy. Since the pancreatic polypeptide response to hypoglycemia is mediated by cholinergic mechanisms, we used this response as an indicator of autonomic neuropathy to determine whether deficient epinephrine and glucagon responses in IDDM could be ascribed to an autonomic defect. The relationships between pancreatic polypeptide, epinephrine, and glucagon responses during insulin-induced hypoglycemia were assessed in 18 patients with IDDM who had no overt evidence of autonomic neuropathy, including normal standard cardiovascular reflex tests, and 11 age-matched nondiabetic subjects. All of the diabetic patients had impaired glucagon responses [19 ± 3 (sem) vs. 96 ± 11 pg/ml, peak increment, P < 0.001]. Ten of the 18 diabetic patients had either impairment of plasma epinephrine or plasma pancreatic polypeptide responses or both to hypoglycemia. Moreover, pancreatic polypeptide responses were significantly correlated with epinephrine responses (r = 0.53, P < 0.003). There was no association between the plasma glucagon response and the epinephrine (r = 0.02, NS), norepinephrine (r = 0.03, NS), or pancreatic polypeptide (r = 0.35, NS) response. Last, there was no correlation between the plasma hormone responses and the cardiovascular reflex test results. Therefore, the association of impaired plasma pancreatic polypeptide responses with impaired plasma epinephrine responses suggests that the impaired epinephrine responses are due to autonomic neuropathy, whereas the dissociation of plasma glucagon responses with both plasma pancreatic polypeptide and epinephrine responses suggests that the impaired pancreatic α-cell response to hypoglycemia is not due to autonomic neuropathy. In addition, the plasma pancreatic polypeptide and epinephrine responses to hypoglycemia appear to be an earlier indicator of underlying autonomic dysfunction than standard cardiovascular reflex tests. Thus, the responses of plasma pancreatic polypeptide and epinephrine to insulin-induced hypoglycemia may be a useful test for the identification of early autonomic neuropathy in IDDM.