Two GABAA receptor subunit-specific antibodies anti-alpha 6 and anti-alpha 1 have been used for elucidating the relationship between the presence of alpha 1 and/or alpha 6 subunits in the cerebellar GABAA receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABAA receptors have alpha 6, whereas 76% of the receptors have alpha 1 as determined by [3H]-muscimol binding. Results show that 42-45% of the receptors having alpha 6 also have alpha 1, whereas 13-15% of the receptors that contain alpha 1 also have alpha 6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both alpha 1 and alpha 6 subunits, a second type has alpha 1 but not alpha 6, and a third type has alpha 6 but not alpha 1 subunits. The results also show that receptors where alpha 1 and alpha 6 subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different alpha subunit. The alpha 1 subunit contributes the high-affinity binding of [3H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [3H]Ro 15-4513. The alpha 6 subunit contributes the diazepam-insensitive binding of [3H]Ro 15-4513, but it does not bind [3H]Ro 15-1788 with high affinity. Thus, in the cerebellar alpha 1- alpha 6 GABAA receptors, there is no dominance of the pharmacology of one alpha subunit over the other.