The presence of allosteric binding sites on recombinant GABAA receptors formed after transfection of human embryonic kidney (HEK) 293 cells with alpha 1-, beta 3-, or gamma 2-subunits, or with various combinations of these subunits, was systematically investigated. From all possible subunit combinations, high affinity [3H]muscimol binding sites were induced in cells transfected with alpha 1 beta 3- or alpha 1 beta 3 gamma 2-subunits only. GABAA receptor associated [3H]flunitrazepam binding sites were induced in cells after transfection with alpha 1 gamma 2- or alpha 1 beta 3, gamma 2-subunits, and [35S]r-butylbicyclophosphorothionate (TBPS) binding sites were found in cells transfected with beta 3-, beta 3 gamma 2-, alpha 1 beta 3-, or alpha 1 beta 3 gamma 2-subunits. Binding of [35S]TBPS could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and 4'-chlorodiazepam (Ro 5-4864) with a potency which differed in cells transfected with beta 3-, beta 3 gamma 2-, alpha 1 beta 3-, or alpha 1 beta 3 gamma 2-subunits. Results obtained indicate that receptors with different subunit composition actually can be formed in HEK cells and exhibit distinct pharmacological properties.