Using immunohistochemistry and in situ hybridization, we have analysed the distribution of vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in hypothalamus of male and female Sprague-Dawley rats under normal and experimental conditions. In most cases there was a good overlap between the distribution of VIP- and PHI-immunoreactive structures. At the median eminence level precapillary arterioles along its lateral aspect were surrounded by dense networks of VIP/PHI-positive fibers, suggesting that these peptides, via their vasodilatory property, may be involved in control of blood flow through portal vessels. Furthermore, a thick VIP/PHI-containing nerve bundle of variable size was observed on the surface of the median eminence in coronal, horizontal and sagittal sections. Also this bundle could be of importance for portal circulation, but VIP/PHI released may reach the anterior pituitary level and play a role in, for example, control of prolactin release. Although different lesions were performed, the origin of the VIP/PHI nerves around lateral blood arterioles and of the bundle is still unclear, but is in all probability peripheral. Within the median eminence of untreated rats only few positive nerve endings were seen in the external layer, but after 48 h hypophysectomy a large number of PHI-immunoreactive fibers could be observed. With regard to cell bodies the suprachiasmatic nucleus contained VIP/PHI-immunoreactive neurons even in untreated rats. After colchicine administration fluorescent cells were in addition seen in several other hypothalamic nuclei, including the parvocellular paraventricular nucleus. After hypophysectomy, with in situ hybridization, VIP mRNA could be demonstrated in magno- and parvocellular neurons of the paraventricular nucleus, whereas in control rats VIP mRNA was undetectable. These results demonstrate that VIP/PHI are present in at least three systems of direct neuroendocrine importance: (1) in nerves related to the blood vessels in the median eminence and presumably involved in control of blood flow through the portal system; (2) in parvocellular paraventricular neurons, presumably related to stress-induced prolactin release; and (3) in magnocellular neurons after certain experimental manipulations.