α-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in α-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of α-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their 1H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced 125I-labeled GI from the Torpedo californica nAChR. 125I-labeled GI(Bpa12) bound to two sites on the receptor (Kd 13 and 1800 nM), and on UV irradiation specifically photolabeled the α, γ and δ subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the α subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at αSer173 and containing the agonist-binding site segment C. In the γ subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at γAla167 and γAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists. © 2006 The Authors.