Structure-function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1-27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1-27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N-terminal or the helical C-terminal domain or a short loop region within the C-terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C-terminal domain of PACAP(1-27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125I-labeled photoreactive analogues specifically label a protein band of M(r) 66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr22 and Lys15 in PACAP (1-27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.