Antecedent hypoglycemia is a primary factor in hypoglycemia-associated autonomic failure, a pathophysiological condition characterized by impaired glucose counterregulatory function. Conventional therapeutic strategies involving administration of intermediate dosage-release formulations of insulin in the management of insulin-dependent diabetes mellitus result in frequent iatrogenic hypoglycemia. This study investigated the neuroanatomical location, direction, and magnitude of CNS neuronal genomic activation by singular versus repeated induction of hypoglycemic bouts of greater than 6 h duration achieved by administration of the intermediate-acting insulin, humulin neutral protamine Hagedorn (NPH). Adult male rats injected subcutaneously with Humulin NPH exhibited robust immunolabeling for the nuclear transcription factor, Fos, in discrete telencephalic, diencephalic, midbrain, and caudal hindbrain loci in a pattern that was not identical to that described for regular insulin. Administration of four doses of insulin on as many days significantly diminished or extinguished Fos immunostaining within the parvocellular hypothalamic paraventricular nucleus, lateral hypothalamic area, dorsomedial hypothalamic nucleus, thalamic paraventricular nucleus, nucleus tractus solitarius, and area postrema, but did not modify labeling of other metabolic loci. However, numbers of Fos-immunoreactivity-positive magnocellular neurons in the hypothalamic paraventricular and supraoptic nuclei were significantly increased after the second and fourth insulin doses, relative to the single-dose group. Concurrent observations of exacerbated hypoglycemia and modified patterns of glucoregulatory hormone secretion after serial injections of intermediate-acting insulin suggest that central mechanisms governing compensatory endocrine responses, specifically glucagon, become habituated to repetitive hypoglycemia of extended duration. Resultant alterations in CNS-islet and -adrenomedullary output and hypothalamic-pituitary-adrenal activity may reflect diminished neuronal activation within one or more of the brain loci characterized here by nonuniform transcriptional activation. The current studies provide a neuroanatomical foundation for further investigation of the neurochemical phenotypes and interconnectivity of functionally adaptive neurons, underlying cellular and molecular mechanisms of diminished or enhanced activation, as well as the impact of these modified cellular responses on glucose counterregulation during administration of intermediate-acting insulin.