gamma-Aminobutyric acid (GABA) receptor was solubilized from synaptic membrane of the rat brain by various detergents. Nonidet P-40, a non-ionic detergent, was found to be an effective solubilizing agent, since it caused no interference on the receptor binding assay, yielded a [3H]muscimol binding protein with a high specific activity and no aggregation, and preserved good stability of the solubilized fraction. Ammonium sulfate precipitation of the solubilized supernatant significantly increased the binding of [3H]muscimol to GABA receptor, possibly by removing heat-stable and small molecular inhibitory substances. The specific [3H]muscimol binding to the soluble fraction obtained by Nonidet P-40 treatment and subsequent ammonium sulfate precipitation, was saturable with KD 13 and 64 nM, and Bmax 3.4 and 1.8 pmol/mg protein, respectively. The enhancement of the [3H]muscimol binding by diazepam as found in synaptic membrane was also detected in the soluble fraction. Molecular weight of the [3H]muscimol binding site was determined by gel filtration on Sephadex G-200 and was calculated to be 270,000 daltons. This value was identical with that of the [3H]flunitrazepam binding site which appeared in the same solubilized fraction. These results indicate that the properties of solubilized GABA receptor are identical to those of membrane-bound GABA receptor. Furthermore, the present results suggest that both GABA and benzodiazepine receptors may reside on the same macromolecule in synaptic membrane.