Evidence for an intrinsic effect of insulin on the central nervous system is accumulating. To test the hypothesis that insulin per se may modulate neuroendocrine counterregulation, hypoglycemia perception, and cerebral function in insulin-dependent diabetes mellitus, we examined 27 patients without any sign of classical autonomic neuropathy or evidence of so-called hypoglycemia unawareness. We used the hyperinsulinemic (0.67 vs. 2.00 mU/kg.min), stepped hypoglycemic (5.6/3.5/2.4/2.0 mmol/L) clamp technique to assess the patient's awareness of and response to equivalent hypoglycemic stimuli under different degrees of physiological hyperinsulinemia (approximately 270 vs. approximately 810 pmol/L) after an overnight euglycemic clamp (5.6 mmol/L). Simultaneously, the patient's cerebral function was assessed from his electrophysiological activity and neuropsychological skills. Higher degrees of physiological hyperinsulinemia caused enhanced neuroendocrine response (adrenaline, P < 0.05; noradrenaline, P < 0.03; GH, P < 0.02; beta-endorphin, P < 0.03; ACTH, P = 0.12; cortisol, P = 0.06; PRL, P = 0.08) and symptom awareness (total symptoms, P < 0.04; autonomic symptoms, P < 0.02; neuroglycopenia symptoms, P < 0.05; sweating, P < 0.05; heart pounding, P < 0.02; trembling, P < 0.01; lack of concentration, P < 0.02) to occur. Deteriorations of electrophysiological activity (middle latency auditory-evoked potentials, P < 0.04; Pa peak latencies, P < 0.05; Pa-V interpeak latencies, P = 0.08) and neuropsychological skills (Stroop test, P < 0.05; trail making, P = 0.12) were more pronounced the higher the insulin level, but at similar blood glucose concentrations. We conclude that insulin-associated modulation of neuroendocrine counterregulation, hypoglycemia perception, and cerebral function may occur in insulin-dependent diabetes mellitus, which indicates an intrinsic effect of insulin on the human brain.