Native GABAA receptors containing different gamma-subunit variants were distinguished immunobiochemically with antisera selectively recognizing the gamma 1-, gamma 2- and gamma 3-subunits. While GABAA receptors containing the gamma 2-subunits were confirmed to be rather ubiquitous in the adult brain, receptors characterized by the gamma 1- or gamma 3-subunit were of low abundance, as shown by immunoprecipitation. The three receptor populations differed strikingly in their benzodiazepine (BZ) site ligand binding profiles. The gamma 3-receptor population displayed reduced affinity for the full agonists clonazepam flunitrazepam and virtually lacked sensitivity to zolpidem. The gamma 1-receptor population displayed low affinity for all benzodiazepine site ligands tested, except for flunitrazepam, and could be differentiated from the gamma 2- and gamma 3-receptors by its low affinity for the inverse agonist beta CCM and its lack of affinity for the partial inverse agonist Ro 15-4513 and the antagonist flumazenil. Since flumazenil antagonizes all major effects of BZ agonists, gamma 1-receptors are not involved in mediating these actions in vivo. In immunopurified receptors, the gamma-subunit variants were found to be assembled with different variants of alpha- and beta-subunits, indicating that not only the gamma 2-subunit gamma 1- and gamma 3-subunits are part of various receptor subtypes. In addition, the gamma 2- and gamma 3-subunits can be co-assembled in native receptors, consistent with the subunit stoichiometry of two alpha-, one beta- and two gamma-subunits proposed previously for recombinant receptors.