Pituitary adenylate cyclase activating polypeptide (PACAP) is structurally similar to vasoactive intestinal polypeptide (VIP). We investigated the characteristics and topographical distribution of [¹²⁵I]PACAP binding sites, compared with those of [¹²⁵I]VIP binding sites in the rat brain. Radiolabeled PACAP and VIP showed highly specific binding to sections at the level of the dorsal hippocampus. The specific binding of [¹²⁵I]PACAP was 10 times higher than that of [¹²⁵I]VIP in hippocampal sections. [¹²⁵I]PACAP binding was scarcely displaced by unlabeled VIP, while [¹²⁵I]VIP binding was effectively displaced by unlabeled PACAP. Therefore, PACAP binding sites may reflect both PACAP specific binding sites and VIP/PACAP binding sites. However, the amount of VIP/PACAP binding sites was negligibly low. Autoradiography revealed that [¹²⁵I]PACAP binding sites were dense in the piriform cortex, diagonal band, accumbens nucleus, anterior part of the striatum, hippocampal formation, habenular nucleus, lateral hypothalamic area, superior colliculus and dorsal raphe nucleus. Moderate to high labeling was observed in the medial septal nucleus, olfactory tubercle, caudal part of the striatum, most parts of the thalamus, supraoptic and periventricular hypothalamic nuclei, central gray, substantia nigra pars compacta, locus coeruleus, pontine reticular nucleus and cerebellum. Distribution pattern was remarkably different from that of [¹²⁵I]VIP binding sites in the hippocampal formation, lateral hypothalamic area, substantia nigra pars compacta, pontine reticular nucleus and cerebellum. The present results suggest that PACAP may have a physiological role in the regulation of the central nervous system.