Data are presented to show that vasoactive intestinal peptide (VIP) is synthesized and secreted by the hypothalamus and anterior pituitary and that it participates in the regulation of pituitary functions. Immunoreactive VIP in the hypothalamus and pituitary is increased following estrogen treatment and adrenalectomy and is reduced in hyperprolactinemic states. The level of VIP mRNA in the hypothalamus is increased during lactation and sexual maturation, while that in the anterior pituitary shows a sexual dimorphism and is increased with estrogen treatment and hypothyroidism. All these findings suggest a physiological regulation of hypothalamic and pituitary VIP gene expression in relation to its potential role as a neuroendocrine hormone. Furthermore, VIP stimulates prolactin (PRL) release at concentrations attainable in the hypophyseal-portal blood. Passive immunoneutralization studies with anti-VIP antisera suggest that endogenous VIP acts at multiple loci in the hypothalamic-pituitary axis to regulate PRL secretion, interacting possibly with other regulators of PRL secretion such as estrogen, serotonin, cholecystokinin, prostaglandins, galanin and oxytocin. Regarding other pituitary functions, although VIP has been shown to release growth hormone, ACTH, and vasopressin in vivo and in vitro, the physiological significance of these findings remains to be determined.