Vasoactive intestinal peptide (VIP) is a prolactin-releasing factor in turkey hens. Membranes from anterior pituitaries of turkey hens were used to characterize VIP receptors. Using HPLC-purified monoiodinated VIP, we found specific VIP receptors in the anterior pituitary glands. Binding was saturable and was time- and temperature-dependent. Scatchard analysis of competitive binding studies indicated two binding sites, a high-affinity binding site (K(d1)) of 6.6 ± 1.8 pM and maximum binding (B(max1)) of 1.52 ± 0.2 pM, and a low-affinity binding site (K(d2)) of 542 ± 200 pM and B(max2) of 15.8 ± 8.0 pM. Binding of VIP to pituitary membranes was specific, as compared to other peptides of the glucagon family. The rank order of potency of the peptides tested was chicken VIP > porcine VIP > peptide histidine isoleucine > secretin > glucagon > growth hormone-releasing factor. Two binding sites were found in all the examined reproductive stages. The lowest binding site levels were found in nonphotostimulated and photorefractory birds, followed by photostimulated birds and layers; highest levels were found in incubating birds. Nest deprivation significantly reduced B(max1) levels without changing hypothalamic VIP content. These results suggest the involvement of the anterior pituitary VIP receptors in the regulation of prolactin secretion from the pituitary gland.