A urotensin II (U-II) peptide analogue containing the photoreactive p -benzoyl-L-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe(6)]U-II bound the receptor expressed in COS-7 cells with high affinity (IC(50) 0.7 nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with (125)I-[Bz-Phe(6)]U-II resulted in the specific formation of a glycosylated (125)I-[Bz-Phe(6)]U-II-U-II receptor complex of 60 kDa. Digestion of the 60 kDa complex with endoproteinase Glu-C generated a fragment of 17 kDa circumscribing the labelled fragment to residues 148-286. Digestion of the ligand-receptor complex with endoproteinase Arg-C produced a short peptide of 4 kDa corresponding to fragments 125-148, 167-192 or 210-233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2 kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met(184) and/or Met(185) of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.