The concentration of human β-endorphin (β_h-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 β_h-EP concentrations in the arcuate nucleus (169 ± 35 pg/100 μ g protein, n = 7) and median eminence (163 ± 32 pg/100 μ g protein, n = 6) were among the highest in the 17 brain areas examined. The immunoreactive β_h-EP in the hypothalamus corresponded to authentic β_h-EP, as determined by gel exclusion chromatography. By chromatography and RIA the β_h-EP concentrations in anterior (1.53 × 10⁵ ±0.51 × 10⁵ pg/100 μ g protein, n = 3) and posterior (1.41 × 10⁵ ± 0.38 × 10⁵ pg/100 μ g protein, n = 5) pituitary were found to be approximately 1,000-fold higher than in hypothalamus. Within the pituitary β_h-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. β_h-EP and adrenocorticotropin (ACTH) were co-localized in the same pituitary cells. The present data represent the first unequivocal localization and quantitation of β_h-EP in human brain and in the separate lobes of the human pituitary.