We previously reported that the injection of neostigmine, an inhibitor of acetylcholinesterase, into the third cerebral ventricle of fasted rats produced hyperglycemia associated with the secretion of epinephrine and norepinephrine. However, the central nervous system site of action of neostigmine by which the plasma catecholamine and glucose concentrations were increased is not known. In this study we injected neostigmine into the ventromedial hypothalamus, lateral hypothalamus, paraventricular hypothalamus, median site of the lateral-preoptic area, lateral site of the lateral-preoptic area, anterior site of the anterior hypothalamic area, mammillary body (posterior mamillary nucleus), and cortex of anesthetized fasted rats and measured the plasma levels of glucose, epinephrine, and norepinephrine. It was found that the ventromedial hypothalamus, lateral hypothalamus, paraventricular hypothalamus, and median site of the lateral-preoptic area were involved in increasing the plasma levels of glucose and epinephrine. From this evidence we conclude that neostigmine acts on selected regions known to be involved in glucoregulation in the hypothalamus to increase the plasma levels of epinephrine and glucose.