GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.