Human embryonic kidney 293 cells transfected with alpha1beta1gamma2, alpha1beta2gamma2, alpha1beta3gamma2, alpha1beta1, alpha1beta2, alpha1beta3, beta3gamma2, or beta3 subunits formed gamma-aminobutyric acidA receptors on the cell surface that could be clustered by rapsyn. In contrast, alpha1, beta1, beta2, or gamma2 subunits, or alpha1gamma2 subunit combinations could not be detected on the surface of transfected cells and could not be clustered by rapsyn. Experiments investigating the ability of rapsyn to cluster chimeras consisting of the N-terminus of the beta3 subunit and the remaining part of the alpha1, beta2 or gamma2 subunits indicated that the intracellular domains of beta1, beta2, beta3 or gamma2 subunits, but not those of alpha1 subunits are able to form sites mediating clustering by rapsyn. These results demonstrate that rapsyn has the potential to cluster the majority of GABA(A) receptor subtypes via beta or gamma2 subunits. Further experiments will have to clarify the physiological importance of this observation.