The effect of hypophysectomy on the number of corticosterone receptor sites was investigated in three rat brain regions and was compared with the effect of long-term adrenalectomy. Subsequently, the effect on receptor capacity was measured after the hypophysectomized rats had received as substitution therapy ACTH1-24 and smaller peptide fragments lacking corticotropic activity. All rats (sham and hypophysectomy) were adrenalectomized 24 h prior to sacrifice for depletion of endogenous adrenal hormones and replacement therapy was discontinued at that time. Extensive perfusion with saline was carried out at the time of sacrifice. 3H-corticosterone binding was measured in cytosol by means of an in vitro assay. 2 weeks after hypophysectomy, the apparent maximal binding capacity (Bmax) of the corticosterone receptor system was increased by 60, 36 and 72% in hippocampus, hypothalamus and septum, respectively. The increase in Bmax in the hippocampus of hypophysectomized rats was 30% higher than the increase in animals adrenalectomized 2 weeks previously. Replacement with ACTH1-24 markedly decreased the binding capacity in all brain regions investigated. Replacement with the behaviorally active ACTH4-10 sequence reduced the number of corticosterone receptor sites in the hippocampus by 21%, while the behaviorally inactive ACTH11-24 sequence was ineffective. Des-glycinamide-arginine-vasopressin was also ineffective. There were no alterations in binding affinity for corticosterone in hippocampal cytosol after the surgical procedures or after the different replacement therapies. It is concluded that the neurotropic ACTH4-10 sequence reduces the number of corticosterone receptor sites in the hippocampus of the hypophysectomized rat. The action of ACTH4-10 was specific for the hippocampus and was not observed in other brain regions or plasma transcortin.