In this study, we investigated the hypothalamic regulatory role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the control of LH and PRL secretion. Overiectomized ewes were surgically prepared with bilateral guide tubes directed at the preoptic area (POA) or medial basal hypothalamus (MBH). After recovery from surgery, PACAP38 (0.1 nmol in 2.5 microliters over 1 h) or vehicle was bilaterally infused into each site in separate trials. Infusion of PACAP38 into the POA had no effect on either LH or PRL secretion. However, infusion of the peptide into the MBH suppressed PRL secretion during the 3-h postinfusion period; the responding animals (n = 9) had injectors located in the arcuate nucleus. In the three nonresponding animals, both injectors were outside the arcuate nucleus. Mean LH concentration, LH pulse frequency, and pulse amplitude were also significantly suppressed, with LH pulsatility declining in seven of eight animals during infusion of the peptide in the MBH. These results suggest that PACAP acts in the arcuate nucleus region of the MBH, and not the rostral POA, to inhibit both LH and PRL secretion.