We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical alpha1-alpha3 gamma-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of alpha1 and alpha3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-alpha1- or anti-alpha3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the alpha1-alpha3 GABAA receptors represented 54.7 +/- 5.0 and 23.6 +/- 3.3% of the alpha3 and alpha1 populations, respectively. Therefore, alpha1 and alpha3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these alpha1-alpha3 GABAA receptors from the cortex constitute a large proportion of the total alpha3 population and a relatively minor component of the alpha1 population. The pharmacological analysis of the alpha1- or alpha3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both alpha1 and alpha3 subunits, with alpha1 and alpha3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-alpha1-anti-alpha3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of alpha1 and alpha3 subunits, associated with beta2/3, and gamma2 subunits. The pharmacological analysis of alpha1-alpha3 GABAA receptors demonstrated that, despite the fact that each alpha subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the alpha1 subunit is pharmacologically predominant over the alpha3 subunit. These results indicate the existence of active and nonactive alpha subunits in the native alpha1-alpha3 GABAA receptors from rat cortex.