The concentration of human β-endorphin (βh-EP) was measured in various hypothalamic nuclei, in extra-hypothalamic brain regions and in the anterior and posterior lobes of the pituitary using a specific radioimmunoassay (RIA). The βh-EP concentrations in the arcuate nucleus (169 ± 35 pg/100 μg protein, n = 7) and median eminence (163 ± 32 pg/100 μg protein, n = 6) were among the highest in the 17 brain areas examined. The immunoreactive βh-EP in the hypothalamus corresponded to authentic βh-EP, as determined by gel exclusion chromatography. By chromatography and RIA the βh-EP concentrations in anterior (1.53 × 105 ±0.51 × 105 pg/100 μg protein, n = 3) and posterior (1.41 × 105 ± 0.38 × 105 pg/100 μg protein, n = 5) pituitary were found to be approximately 1,000-fold higher than in hypothalamus. Within the pituitary βh-EP was localized throughout the anterior lobe, in the pars intermedia and in that part of the posterior lobe nearest the pars intermedia, as judged by immunocytochemistry. Dense immunocytochemical staining was found along the perimeter of many blood vessels. βh-EP and adrenocorticotropin (ACTH) were co-localized in the same pituitary cells. The present data represent the first unequivocal localization and quantitation of βh-EP in human brain and in the separate lobes of