We adapted an uncommon peroxidase substrate, ortho-dianisidine, to double immunostaining methodology in order to analyze the degree of neuronal coexistence of pro-opiomelanocortin-derived peptides and to compare the distribution of neurons containing beta-endorphin immunoreactivity (ir) with those containing enkephalin-ir and dynorphin-ir in the medial basal hypothalamus. Double immunostaining demonstrated co-localization of beta-endorphin-ir, adrenocorticotropic hormone-ir, and gamma 3-melanocyte stimulating hormone-ir in medial basal hypothalamus (MBH) neurons. However, while all perikarya containing beta-endorphin-ir invariably contained adrenocorticotropic hormone-ir, not all of these cell bodies contained gamma 3-melanocyte stimulating hormone-ir. In contrast, separate neuronal cell bodies containing beta-endorphin-ir, enkephalin-ir, and dynorphin-ir were distributed throughout the rostral-caudal extent of the MBH. Fibers containing enkephalin-ir closely surrounded cell bodies containing beta-endorphin-ir, suggesting axosomatic contacts between these two opioid peptidergic neuronal populations.