Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of α-conotoxin MI and L-benzoylphenylalanine (Bpa) analogs of α-conotoxin GI were synthesized. All these compounds, similarly to native α-conotoxins, completely displaced the radioiodinated MI or GI from the membrane-bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated α-bungarotoxin (αBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross-linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native α-conotoxins or αBgt. Both equilibrium binding of benzophenone-derivatized α-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for α-conotoxins and αBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable α-conotoxins and (c) enhancement of benzophenone-derivatized α-conotoxins cross-linking at additional (physostigmine-related) sites by αBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.