Pituitary adenylate cyclase activating peptide (PACAP) tested as PACAP-(1-38)NH2 and PACAP-(1-27)NH2 and vasoactive intestinal polypeptide (VIP) were compared for their capacity to discriminate between high- and low-affinity VIP-preferring receptors that coexist in rat liver plasma membranes. This capacity was evaluated by the ability to 1) inhibit 125I-labeled-PACAP-(1-27)NH2, 125I-labeled-VIP, and 125I-labeled-helodermin binding and 2) to activate adenylate cyclase. PACAP-(1-27)NH2 bound specifically and reversibly to three classes of binding sites, as revealed by analysis of binding curves. On high-affinity VIP receptors (tested specifically by [125I]-helodermin binding), PACAP-(1-38)NH2 showed lower affinity than PACAP-(1-27)NH2 and VIP itself. On low-affinity VIP receptors, PACAP-(1-27)NH2 and -(1-38)NH2 showed similar modest affinity that was slightly higher however than that of VIP. For a third specific class of PACAP receptors (20% of PACAP receptors not recognized by VIP), PACAP-(1-38)NH2 showed higher affinity than PACAP-(1-27)NH2. Both PACAPs stimulated rat liver adenylate cyclase with the same low efficacy as VIP but with an affinity even greater [half-maximal effective concentration (EC50) 0.02 nM] than that of VIP (EC50 0.05 nM).