The effects of various physical, chemical and enzymatic treatments of rat brain membranes were investigated with respect to 125I-labeled alpha-bungarotoxin ([125I]alpha BuTx) and [3H]quinuclidinylbenzilate ([3H]QNB) binding. Binding appeared relatively stable to autolysis, mechanical shearing, freeze-thawing, and divalent cation addition (Sr2+) or removal (EGTA, EDTA). Binding for [125I]-alpha BuTx was slightly reduced by trypsin digestion of the membranes while both [125I]alpha BuTx and [3H]QNB binding were reduced by phospholipase A2 digestion (Crotalus adamantus phospholipase A2 and beta-bungarotoxin). Treatment of the membranes with the disulfide reducing agent, dithiothreitol, resulted in additional [125I]alpha BuTx binding but showed little effect on [3H]QNB binding. Binding of the cholinergic agonists, nicotine and carbamylcholine, was studied by observing their concentration-dependent ability to inhibit [125I]alpha BuTx and [3H]QNB binding, respectively. Membrane sulfhydryl group reduction and endogenous cation removal by EGTA or EDTA resulted in a lowered affinity for nicotine bindng. Alkylation of membranes with N-ethylmaleimide resulted in an increase in carbamylcholine affinity. Other treatments had little or no effect on nicotine or carbamylcholine binding.