Several photoaffinity derivatives of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana have been prepared. After reaction of the 125I-labeled derivatives with the nicotinic acetylcholine receptor from electric organ, the a-subunit of the nAChR is almost exclusively labeled by the derivative carrying the photoactivatable group in position Lys46. In contrast to this, a reactive group at Lys26 predominantly labels the γ- and δ-subunits, while the α- and β-subunits incorporate much less radioactivity. Competition experiments with D-tubocurarine show that the γ-subunit is labeled when this derivative occupies the high affinity d-tubocurarine-binding site, while the δ-subunit is labeled by the toxin bound at the low-affinity d-tubocurarine site. A model is discussed for the orientation of different loops of the toxin molecules in the binding site for agonists and competitive antagonists. © 1992, American Chemical Society. All rights reserved.