In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of ¹²⁵I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in ~100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity ¹²⁵I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (K_d) and maximal binding capacities (B_max) were as follows: K_{d H} = 66.9 pM, B_{max H} = 101 fmol/mg; K_{d L} = 2.18 nM, B_{max L} = 580 fmol/mg protein. The binding of ¹²⁵I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5'-O-(3-thiotriphosphate) (GTPS). GTPS or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had ~100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an ~70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly G_s-coupled, receptors in DMP synaptosomes and CM membranes.