The widespread 28-amino acid neuropeptide vasoactive intestinal peptide (VIP) exerts its many biological effects through interaction with serpentine class II G protein-coupled receptors named VPAC receptors. We previously provided evidence for a physical contact between the side chain at position 22 of VIP and the N-terminal ectodomain of the hVPAC1 receptor (Tan, Y. V., Couvineau, A., Van Rampelbergh, J., and Laburthe, M. (2003) J. Biol. Chem. 278, 36531-36536). We explored here the contact site between hVPAC1 receptor and the side chain at position 6 of VIP by photoaffinity labeling. The photoreactive para-benzoyl-l-Phe (Bpa) was substituted for Phe(6) in VIP resulting in [Bpa(6)]-VIP, which was shown to be a hVPAC1 receptor agonist in Chinese hamster ovary cells stably expressing the recombinant receptor. After obtaining the covalent (125)I-[Bpa(6)-VIP].hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis. The data demonstrated that (125)I-[Bpa(6)-VIP] were covalently attached to the short 104-108 fragment within the N-terminal ectodomain of the receptor. The data were confirmed by creation of a receptor mutant with new CNBr cleavage site. In a three-dimensional model of the receptor N-terminal ectodomain, this fragment was located on one edge of the putative VIP-binding groove and was adjacent to the fragment covalently attached to the side chain at position 22 of VIP. Altogether these data showed that the central part of VIP, at least between Phe(6) and Tyr(22), interacts with the N-terminal ectodomain of the hVPAC1 receptor.