Previous studies have shown that the pattern and degree of 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) photoincorporation into the nicotinic acetylcholine receptor (nAChR) can be used as a sensitive measure of nAChR conformation. Upon desensitization by prolonged exposure to agonists, certain drugs and detergents, or reconstitution into desensitizing lipids, the levels of [125I]TID incorporation into the subunits of the nAChR are dramatically reduced. In this study, we characterized the effects of the snake venom proteins alpha-bungarotoxin and alpha-cobrotoxin, as well as the smaller antagonists tubocurarine and gallamine, on [125I]TID incorporation into the subunits of both partially-purified nAChR in native lipids, or affinity-purified nAChR reconstituted into different combinations of lipids. Unlike all other compounds previously tested, alpha-bungarotoxin and alpha-cobrotoxin reproducibly increased the level of [125I]TID incorporation into all four subunits of nAChR reconstituted into dioleoylphosphatidylcholine, dioleoylphosphatidic acid and cholesterol. Gallamine had little or no effect on [125I]TID incorporation at any concentration tested (0.1 microM-5 mM). Tubocurarine had no effect on [125I]TID incorporation at low concentrations, but at higher concentrations reduced the level of [125I]TID labeling. The snake venom proteins may shift the population of nAChR, which exists as a mixture of resting state and desensitized conformations, entirely to the resting state. However, the binding of the snake venom toxins does not appear sufficient to induce the resting state conformation in nAChR which have been desensitized by other means, such as solubilization in desensitizing detergents or reconstitution in densitizing lipids.