In investigating the agonist binding site of the human brain cholecystokininB receptor (CCKBR), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (KD = 1.2 nM) to the CCKBR and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCKBR. Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a protein core of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequent chemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified by Edman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. The position of the radioactive probe within the identified peptide was determined using combined tandem electrospray mass spectrometry and peptide mapping. The probe was covalently attached within the sequence L52ELAIRITLY61 that represents the transition between the N-terminal domain and predicted transmembrane domain 1. Using this interaction as a constraint to orientate the ligand within the putative receptor binding site, a model of the CCK-8s-occupied CCKBR was constructed. The hormone was found to be placed in a binding pocket built from both extracellular and transmembrane domains of CCKBR with its N-terminus mainly interacting with residues Arg57 and Tyr61.