AbstractPurifiedTorpedo nobilianaelectric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)‐stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3‐H]‐phencyclidine ([3‐H]‐PCP) binding to the channel sites, acting as a weak agonist. The activation of [3‐H]‐PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb‐activated [3‐H]‐PCP binding with Kivalues of 10, 20, and 1,600 μM, respectively. The inhibition was mixed competitivenoncompetitive in nature. The characteristic response of CRPVF to carb‐stimulated [22‐Na] influx was inhibited by the three carbamates, with IC‐50 values of 6,50, and 1,000 μM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb‐stimulated [22‐Na]influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125‐I]‐α‐ bungarotoxin ([125‐I]‐α‐BGT) with the following decreasing order of potency: Neo