The hypothesis that vasoactive intestinal peptide (VIP) functions as a hypothalmic prolactin (PRL)-releasing peptide in the turkey was tested by determining the effects of hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade on hypothalamic extract (HE)-induced PRL secretion from dispersed anterior pituitaries. Incubation of cells with porcine VIP (pVIP; 0.5 or 10 nM) significantly stimulated PRL secretion. This effect was inhibited in a dose-related manner by 1-hr preincubation of pVIP with a VIP antisera ( A S; 1:500-1:50,000). Likewise, HE (0.3 equivalent)-stimulated PRL secretion was inhibited by preincubation with VIP A S (P < 0.0001). A 96-98% reduction in PRL secretion was obtained from cells cultured with HE, that was previously incubated with 1 500 dilution of antiserum. Pretreatment of pituitary cells for 15 min with [4Cl-d-Phe6,Leu17] VIP, a VIP receptor antagonist (10-5 M), significantly depressed the PRL response to 0.5 nM VIP (9.9 ± 0.5 μg/500,000 cells vs 4.9 ± 0.1 μg/500,000 cells; 22.4 ± 0.9 μg/500,000 cells vs 14.7 ± 0.4 μg/500,000 cells) or 0.3 eq HE (8.8 ± 0.6 μg/500,000 cells vs 5.2 ± 0.2 μg/500,000 cells; 15.3 ± 0.3 μg/500,000 cells vs 8.2 ± 0.2 μg/500,000 cells). These results suggest that hypothalamic stimulation of PRL secretion appears to be mediated by receptors specific for VIP and that VIP is an endogenous hypothalamic PRL-releasing peptide in the turkey.