The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel made up of homologous subunits (a₂bgd) associated around a central axis that is the ion channel.  The nAChR contains two binding sites for agonists/competitive antagonists, located at a-g and a-d subunit interfaces.  Previous affinity labeling and mutational studies have identified amino acids in 3 discontinuous regions of a-subunit primary structure (Loops A-C) and 3 regions of g- (or d-) subunit (Loops D-F) that contribute to the acetylcholine (ACh) binding sites.  To define the orientation of drugs bound within the ACh sites, we synthesized two novel photoactivatable choline esters ([³H]4-benzoylbenzoyl-choline (Bz₂choline) and [³H]4-(1-azi-2,2,2-trifluoromethyl)benzoylcholine (ATFBcholine) and studied their interactions with Torpedo nAChRs.  Both drugs act as nAChR competitive antagonists, and each binds with the same affinity to the two ACh sites (Bz₂choline, K_eq = 5 mM; ATFBcholine, K_eq = 10 mM).  When equilibrated with Torpedo nAChR-rich membranes, irradiation at 365 nm resulted in pharmacologically specific incorporation of [³H]Bz₂choline into g- and d-subunits, but not a-subunit, while [³H]ATFBcholine was incorporated into a- as well as g- and d-subunits.  Photolabeled amino acids were identified by sequence analysis of ³H-peptides isolated from nAChR subunits after enzymatic digestion, SDS-PAGE and HPLC.  [³H]Bz₂choline reacted solely with an amino acid in Loop E (gLeu-109/dLeu-111) while [³H]ATFBcholine reacted with that amino acid as well as amino acids in a-subunit Loop C.  These results establish that when choline esters of benzoic acid are bound, the para-substituent is selectively oriented and in proximity to amino acids in Loop E in g/d-subunit and Loop C in a-subunit.