This study was designed to assess effects of insulin-induced hypoglycemia of plasma and cerebrospinal fluid (CSF) levels of immunoreactive (ir) β-endorphins, adrenocorticotropin (ACTH), cortisol, norepinephrine, insulin, and glucose in the conscious, overnight fasted dog. Dogs received either an intravenous infusion of saline or insulin (5 mU/kg/min) for 3 h. Infusion of saline alone in conjunction with acute sampling of CSF caused no measurable perturbations of glucose homeostasis. Insulin infusion caused a 60% drop in both plasma and CSF glucose. Plasma levels of ir-β-endorphins, ACTH and cortisol rose markedly. CSF levels of ir-β-endorphins and ACTH also increased. While the magnitude of the increase was smaller than that in the plasma, it was greater than would be expected if crossover of the peptides from the plasma were the sole source of the increase. Hypoglycemia also induced elevations in CSF cortisol and insulin. In addition, there was a 45% decrease in CSF norepinephrine in spite of large elevations of norepinephrine in the plasma. We conclude that hypoglycemia is associated with marked changes in central as well as peripheral levels of neuroendocrine factors. The importance of these changes in mediating acute and long-term responses to hypoglycemia remains to be established.