The influence of adrenalectomy and corticosterone substitution was investigated on Leu-Phe cleaving endopeptidase activity and on the levels of γ-endorphin and β-endorphin in the pituitary gland and the brain. The enzyme activity was quantitated by a specific radiometric assay based on the cleavage of the Leu17-Phe18 bond in a NH2 and COOH-terminally protected synthetic substrate wich was analogous to β-endorphin-(15–19). This activity may mimick the formation of γ-endorphin. β-Endorphin and γ-endorphin were measured by specific radioimmunoassays. After 14 days of adrenalectomy enzyme activity had increased in anterior (15%) and neurointermediate lobes of the pituitary gland (30%), hypothalamus (25%), and liver (15%). This increase was prevented when the adrenalectomized animals were subjected to chronic corticosterone substitution by subcutaneous implantation of a pellet of 100 mg. Extirpation of only the adrenal medulla did not affect the Leu-Phe cleaving activity. Enzyme activity in the septum, hippocampus, and cerebellum had not changed after adrenalectomy. Determination of immunoreactive levels of γ- and β-endorphins showed that in the anterior pituitary gland γ- and β-endorphins had increased by 275 and 300%, respectively, 14 days after adrenalectomy. No significant changes were observed in endorphin levels of the intermediate lobe of the pituitary gland, hypothalamus, hippocampus, and septum. The results indicate that Leu-Phe cleaving endopeptidase activity is sensitive to glucocorticoids in tissues containing proopiomelanocortin-producing cells, i.e., anterior and neurointermediate pituitary gland and the hypothalamus. In the anterior pituitary gland it is correlated with the levels of γ- and