β-Endorphin and its precursors, β-lipotropin and pro-opiocortin, in the pituitary and hypothalamus of the rat, were studied by means of radioimmunoassays with antisera to β-endorphin, β-melanocyte stimulating hormone and adrenocorticotropin. Gel filtration chromatography in combination with radioimmunoassay revealed the existence of β-endorphin, β-lipotropin and pro-opiocortin in all the tissues studied, with the highest concentration in the posterior-intermediate lobe of the pituitary gland. Great variation was observed in the relative amounts of these three β-endorphin-like immuno-reactive peptides in different regions. The ratio of β-endorphin to β-lipotropin is highest in the hypothalamus, is less in the posterior intermediate pituitary lobe and lowest in the anterior pituitary lobe. A β-melanocyte-stimulating hormone-like immunoreactive peptide, which is larger than porcine β-melanocyte-stimlating hormone and smaller than β-lipotropin, was detected in the posterior-intermediate lobe but not in the anterior lobe of the pituitary. The results suggest that the pro-opiocortin may act mainly as a precursor of adrenocorticotropin in the anterior pituitary and that β-endorphin may act mainly in the intermediate lobe of the pituitary where there is conversion of β-lipotropin to β-endorphin and the β-melanocyte-stimulating hormone-like immunoreactive peptide. © 1979.