Vasoactive intestinal polypeptide (VIP) is produced by neurons in the rat hypothalamic paraventricular nucleus (PVN) and may have an important role as a prolactin-releasing factor. Recent work from our laboratories has shown that thyroid hormone regulates the content of VIP and VIP mRNA in the rat anterior pituitary, but its effect on VIP in the PVN is not known. To determine whether thyroid hormone alters VIP biosynthesis in the PVN, we studied the effect of hypothyroidism on the content of immunoreactive (IR)-VIP and VIP mRNA in PVN neurons using histochemical techniques. By immunocytochemistry, only scattered IR-VIP fibers were present in the PVN of control animals whereas IR-VIP perikarya and fibers were present in hypothyroid rats. By in situ hybridization histochemistry, no labeled neurons were recognized in the PVN in control animals whereas PVN neurons were labeled in hypothyroid rats. These findings raise the possibility that hypothyroidism exerts negative feedback regulation on VIP-producing neurons in the PVN and suggest that this may be important to modulate the stimulatory effects of VIP on anterior and/or posterior pituitary function.