The association of neuropeptide-Y (NPY)-immunoreactive (IR) axon terminals with TRH-synthesizing neurons in the rat hypothalamic paraventricular nucleus (PVN) has been studied. Immunocytochemical single and double labeling studies were performed at both light and electron microscopic levels using antiserum to NPY and, as a marker of TRH-containing neurons, antisera recognizing the N-terminal flanking peptides of the TRH prohormone, prepro-TRH-(25-50) and prepro-TRH-(53-74). At the light microscopic level, a diffuse group of TRH-IR cell bodies were observed in the anterior parvocellular subdivision of the PVN and became more numerous and densely clustered in the medial and periventricular parvocellular subdivisions. NPY-IR fibers were observed to innervate all subdivisions of the PVN, but were particularly dense in the anterior, medial, and periventricular parvocellular subdivisions of the nucleus, where they appeared to contact TRH-synthesizing perikarya and neuronal processes. At the ultrastructural level, numerous NPY-IR axon terminals containing labeled vesicles were either tightly juxtaposed to TRH-producing neurons or seen to establish both symmetric and asymmetric synaptic contacts with TRH-containing cell bodies and dendrites. Some NPY-IR axon terminals also established synaptic contacts with unlabeled PVN perikarya and processes or were found in close apposition to blood vessels. These data provide a morphological basis to suggest NPY-mediated neuroendocrine regulation over the biosynthesis and/or secretion of TRH in the PVN. Reports of the colocalization of NPY and catecholamines in the same axon terminals raises the possibility of a potential interaction between NPY and catecholamines to influence TRH neurons in the PVN. Morphological evidence for synaptic interactions between NPY-IR axon terminals and non-TRH-containing neurons in the PVN further suggests that this peptide may influence other neuroendocrine systems.