Two bioactive products of pituitary adenylate cyclase-activating polypeptide (PACAP) prohormone have been isolated from ovine hypothalamus: PACAP38 with 38 residues and PACAP27 corresponding to the N-terminal 27 residues of PACAP38. Immunocytochemical and RIA results support the existence of PACAP in the rat brain, posterior pituitary, and various peripheral tissues. Furthermore, high affinity PACAP-binding sites have been detected in the rat brain, anterior pituitary, and cultured astrocytes which differ from those in lung, liver, and cultured mouse splenocytes. In the present study additional rat tissues were examined to elucidate the location and characteristics of PACAP-binding sites using [125I] PACAP27 with conventional methods of receptor autoradiography and RRA. Binding specificity was established by displacement with unlabeled PACAP27 or a related peptide, vasoactive intestinal polypeptide (VIP). PACAP27-binding sites were localized autoradiographically in the testis, epididymis, adrenal gland, lung, liver, prostate gland, and seminal vesicle; binding sites were not detected in the heart, kidney, or thymus. In the testis and epididymis, a PACAP27-binding site was localized on germinal cells and in the adrenal gland on medullary chromaffin cells. Excess VIP did not displace PACAP27 binding localized in these three tissues. A site with a greater affinity for PACAP27 than for VIP was detected in adrenal gland and epididymis, characteristic of a site recognized previously in hypothalamus, anterior pituitary, and cultured astrocytes. The PACAP-specific site was more abundant in these tissues than a second site to which PACAP27 and VIP bound with similar affinities. Accordingly, the first site has been named type I. In lung, liver, prostate, and seminal vesicle, VIP displaced PACAP27 binding localized autoradiographically. Lung and liver contained an abundant site to which PACAP27 and VIP bound with similar affinities. This binding site, measured previously in lung, liver, and cultured splenocytes, may be shared by PACAP and VIP and has been named type II. Taken together, these data support the existence of two high affinity binding sites for PACAP with different tissue distribution.