Evidences indicate the existence of two homologous and/or heterologous alpha subunits coassembled in a single gamma-aminobutyric acid type A (GABA(A)) receptor. However, it is unknown whether both or only one of the coassembled alpha subunits display benzodiazepine binding sites. Thus, we have investigated the association between alpha1 and alpha5 subunits and the pharmacological properties of these GABA(A) receptors from rat hippocampus. The association between alpha1 and alpha5 subunits was demonstrated by immunoblot of the anti-alpha1 or -alpha5 immunoaffinity-purified receptors and by double immunopurification by anti-alpha1 and -alpha5 columns in series. The benzodiazepine binding properties of the immunoprecipitated receptors indicated the existence of pharmacologically active and inactive alpha subunits. The anti-alpha5 immunoprecipitated receptors displayed exclusively low-affinity binding sites for both Cl218,872 (K(i) = 0.81 +/- 0.15 microM) and zolpidem (K(i) = 5.0 +/- 3.0 microM), in spite of the association between alpha1 and alpha5 subunits. The anti-alpha1 immunoprecipitated receptors displayed both high- and low-affinity binding sites for both ligands (K(i)s = 47.5 +/- 5.2 nM and 0.7 +/- 0.06 microM for Cl218,872 and 25.0 +/- 7.0 nM, 415 +/- 200 nM and 9. 3 +/- 3.0 microM for zolpidem). Therefore, the alpha5 subunit, when coassembled with alpha1 subunit, should be pharmacologically predominant. This hypothesis was probed by immunoprecipitation of the photoaffinity-labeled receptors and by anti-alpha1 and -alpha5 double immunopurified receptors. The alpha1-alpha5 double immunopurified receptors displayed a single low-affinity binding site (K(i) = 908 +/- 105 nM) for Cl218,872, undetectable [(3)H]zolpidem binding activity, and similar [(3)H]flumazenil and [(3)H]L-655,708 binding activity (0.10 +/- 0.01 and 0.09 +/- 0.02 pmol/20 microliters of anti-alpha5 immunobeads, respectively). Thus, the native GABA(A) receptors containing alpha1 and alpha5 subunits have only one alpha subunit pharmacologically active displaying alpha5 binding properties.