A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the δ subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled δ subunit. The sequence of this peptide, δ-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of α- neurotoxin, finding the cross-link at αAla-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the α- neurotoxin binding site is situated at least ≃40 Å from the extracellular surface of the AChR, proximal to the channel axis.