Immunoreactive fibers and varicosities in the hypothalamic paraventricular nucleus (PVN) were examined by light- and electronmicroscopy, following treatment of brain slices with specific antibodies to adrenocorticotropin (ACTH), beta-endorphin (beta-End) and alpha-melanotropin (alpha-MSH) peptides. In an attempt to provide a more precise, quantitative definition of the densities of immunoreactive elements, sections were analyzed by computer based image-analysis techniques. Fibers and varicosities immunostained with the 3 different antibodies displayed an identical distribution pattern throughout the nucleus suggesting that they are parts of the same, arcuate pro-opiomelanocortin (POMC) neuron system. Although immunoreactive varicosities were found all over the PVN, it was possible to identify a characteristic, density distribution pattern. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites. The majority of immunoreactive elements were found in the dorsal parvo- and caudal magnocellular subdivisions which give rise to long projections to the lower brainstem. Moderate density of POMC neural elements was observed in the anterior and medial (ventral portion) parvocellular subdivisions which project to the external zone of the median eminence. Only a few, widely scattered immunostained varicosities are found in the medial and lateral magnocellular subdivisions which project to the neurohypophysis. A combined lesion and immunocytochemical approach has shown that the bulk of the afferent neuronal input from arcuate POMC cells enters the PVN from a ventral direction.