To identify a parathyroid hormone-binding component in renal membranes, a biologically active photoaffinity radioligand of parathyroid hormone was designed using chemical strategies based on the experience of earlier structure-activity studies. The sulfur-free, oxidation-resistant, synthetic analogue of bovine parathyroid hormone (bPTH), (Nle-8,Nle-18,Tyr-34) bPTH-(1-34) amide, was radiolabeled with /sup 125/iodine. Two photolabile moieties, 4-fluoro-3-nitrophenyl azide and N-succinimidyl-6(4' -azido-2' -nitrophenyl amino)-hexanoate, were separately conjugated to iodinated peptide under conditions favoring coupling through lysine side chains rather than the NH/sub 2/-terminal amino group. Concurrent studies established that a photolabile analogue was fully active in the renal adenylate cyclase assay. After incubation of both photolabile analogues in darkness with renal membranes in the presence or absence of competing hormone, exposure to light covalently linked the analogues to membrane components. Membranes were then solubilized, reduced, and fractionated on sodium dodecyl sulfate poly- acrylamide gels. Radioautographs of the gels showed four to six labeled membrane components for each analogue in the absence of competing hormone, but only one band failed to label in the presence of competing hormone. This band labeled in the presence of inactive synthetic fragments of parathyroid hormone, and was not identified in membrane preparations derived from a renal cell line that is notmore » parathyroid hormone-responsive. The migration of the band (M/sub r/ approx. = 70,000) was identical regardless of the photolabile analogue employed. These studies provide a technique useful for further study of the interactions of parathyroid hormone and receptor in various target tissues and for receptor purification and characterization.« less