Neuroendocrine regulatory peptide (NERP)-3, derived from the neurosecretory protein VGF (non-aconymic), is a new biologically active peptide identified through peptidomic analysis of the peptides secreted by an endocrine cell line. Using a specific antibody recognizing the C-terminal region of NERP-3, immunoreactive (ir)-NERP-3 was identified in acid extracts of rat brain and gut as a 30-residue NERP-3 with N-terminal pyroglutamylation. Assessed by radioimmunoassay, ir-NERP-3 was more abundant in the brain, including the posterior pituitary (PP), than in the gut. Immunohistochemistry demonstrated that ir-NERP-3 was significantly increased in the suprachiasmatic nucleus, the magnocellular division of the paraventricular nucleus, and the external layer of the median eminence, but not in the supraoptic nucleus, after dehydration. The immunoreactivity was, however, markedly decreased in all of these locations after chronic salt loading. Intracerebroventricular administration of NERP-3 in conscious rats induced Fos expression in a subset of arginine vasopressin (AVP)-containing neurons in the supraoptic nucleus and the magnocellular division of the paraventricular nucleus. On in vitro isolated rat PP preparations, NERP-3 caused a significant AVP release in a dose-related manner, suggesting that NERP-3 in the PP could be an autocrine activator of AVP release. Taken together, the present results suggest that NERP-3 in the hypothalamo-neurohypophyseal system may be involved in the regulation of body fluid balance.