The potency of 30 benzodiazepine binding site ligands from 14 different structural classes for inhibition of [3H]Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate) binding to human embryonic kidney (HEK) 293 cells transiently transfected with alpha 4 beta 3 gamma 2S or alpha 1 beta 3 gamma 2S subunits of GABAA receptors was investigated. Most of these compounds were unable to significantly inhibit [3H]Ro 15-4513 binding to alpha 4 beta 3 gamma 2S receptors under conditions where they potently inhibited binding to alpha 1 beta 3 gamma 2S receptors. Nevertheless, compounds from four different structural classes were identified which exhibited a high affinity for alpha 4 beta 3 gamma 2S receptors. Variation of the structure of these compounds could lead to new ligands selectively interacting with alpha 4 beta 3 gamma 2S receptors. Compounds interacting with alpha 4 beta 3 gamma 2S receptor were also able to inhibit [3H]Ro 15-4513 binding to receptors consisting of alpha 4 gamma 2S subunits with comparable potency. These results support the conclusion that the alpha subunit is a major determinant of the benzodiazepine binding site properties of GABAA receptors containing alpha and gamma subunits.