Functional coassembly of gamma-aminobutyric acid (GABA)C rho1 subunits with GABAA (alpha1, beta2, and gamma2S) or glycine (alpha1, alpha2, and beta) subunits was examined using two-electrode voltage-clamp recordings in the Xenopus laevis oocyte expression system. To facilitate this study, we took advantage of the unique gating and pharmacological properties of two mutant rho1 subunits, rho1(T314A) and rho1(T314A/L317A). When the rho1(T314A) subunit was coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, GABA response properties were different from those of homomeric rho1(T314A) receptors. Additionally, the sensitivity of heteromeric rho1(T314A) and gamma2S receptors to picrotoxinin (PTX) blockade of GABA-evoked responses was altered compared to that of homomeric rho1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when rho1(T314A) subunits were coexpressed with wild-type rho1 subunits. When rho1(T314A/L317A) subunits were coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric rho1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA rho1(T314A/L317A)/gamma2S heteromeric receptors displayed an additional component. Coinjection of wild-type rho1 with gamma2S cRNAs at a ratio of 1 : 1 resulted in a > 10-fold reduction in GABA-evoked current. Furthermore, coexpression of wild-type rho1 and gamma2S subunits was found to shift the GABA dose-response curve. Our results provide functional evidence that the GABAC rho1 subunit can coassemble with the GABAA gamma2S subunit, and, at least in its mutated form, rho1 can also form heteromeric receptors with glycine alpha1 or alpha2 subunits in vitro.