Activity of the magnocellular neurons that synthesize vasopressin and oxytocin in the paraventricular and supraoptic nuclei of the hypothalamus can be modulated by local release of neuromediators within the nuclei. Among the bioactive peptides that may play autocrine or paracrine roles in this system is prolactin (PRL). Paraventricular and supraoptic neurons express PRL mRNA and contain and secrete PRL-like proteins of 23 and 14 kDa. We investigated the localization of PRL receptors in vasopressinergic and oxytocinergic magnocellular neurons using dual-label immunofluorescence. The results demonstrate that both vasopressin- and oxytocin-immunoreactive cells of the paraventricular and supraoptic nuclei contain the PRL receptor. In addition, we investigated the possible regulation of vasopressin secretion by PRL using hypothalamo-neurohypophyseal explants in culture. The results show that PRL and a 16 kDa N-terminal fragment of the hormone that is analogous to the neurohypophyseal 14-kDa PRL fragment stimulate the release of vasopressin. Together, these findings support the hypothesis that vasopressinergic and oxytocinergic neurons of the magnocellular secretory system are regulated directly by various isoforms of PRL via autocrine/paracrine mechanisms.