A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.