Human embryonic kidney 293 cells transiently transfected with alpha 4-, beta 3- and gamma 2-subunits of gamma-aminobutyric acidA (GABAA) receptors from the rat exhibited specific high affinity binding sites for [3H]muscimol, [3H]Ro 15-4513 and [35S]t-butylbicyclophosphorothionate (TBPS). Bmax values obtained, however, were dramatically different for these compounds. In addition, GABA was able to inhibit only 20% of specific [35S]TBPS binding to membranes from alpha 4 beta 3 gamma 2-transfected cells. In order to investigate possible receptor heterogeneity, receptors were extracted from alpha 4 beta 3 gamma 2-transfected cells and were fractionated by chromatography on an anti-gamma 2-, followed by an anti-alpha 4- and an anti-beta 3-immunoaffinity column. Western blot analysis of the column eluates indicated the separate existence of GABAA receptors consisting of alpha 4 beta 3 gamma 2-, alpha 4 beta 3- or beta 3-subunits in alpha 4 beta 3 gamma 2-transfected cells. This, and the finding that only alpha 4 beta 3 gamma 2- but not alpha 4 beta 3- or beta 3-receptors possess high affinity binding sites for all three radiolabeled ligands investigated, combined with the observation that [35S]TBPS binding to receptors consisting of beta 3-subunits cannot be inhibited by GABA, can explain most of the binding data obtained. The present results suggest an inefficient assembly of gamma 2- with alpha 4- and/or beta 3-subunits under the conditions used, and indicate that recombinant receptors expressed in HEK cells are not necessarily homogeneous.