The nicotinic acetylcholine receptor (nAChR) antagonist alpha-bungarotoxin (alpha-Btx) binds to two different classes of high affinity binding sites from the Drosophila central nervous system. We have used the bivalent reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to cross-link 125I-alpha-Btx (M(r) = 8 kDa) to Drosophila head membranes. Upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), one major adduct of M(r) approximately 50 kDa was identified, suggesting that a 42 kDa polypeptide binds the toxin. Adduct formation was inhibited by other cholinergic ligands. Detergent-solubilized receptor complexes containing the cross-linked products were immunoprecipitated by antisera against two nAChR subunits previously identified by molecular cloning, the ALS and ARD proteins, suggesting that the 42 kDa toxin binding polypeptide constitutes a component of the previously described class 1 alpha-Btx binding site.