The pharmacological specificity of the binding of 125I-labeled alpha-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the alpha-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1 - 10(-8) M. Nicotine and acetylcholine iodide at concentrations of 10(-5) M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10(-4) M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10(-4) M. These data support the isolation of a partially purified nicotinic acetylcholine receptor protein.