In the central nervous system, receptors for γ-aminobutyric acid (GABA) are responsible for inhibitory neurotransmission. Anatomical and electrophysiological studies indicate that GABAC receptors are composed of ρ subunits. While the ρ1 subunit of various species forms homooligomeric receptors with GABAC-like properties, molecular cloning has identified additional ρ subunits whose functional role is unclear. By RT-PCR, we demonstrated that ρ1 expression is primarily restricted to the retina, whereas the ρ2 subunit was present in all brain regions tested. Transfection of HEK-293 cells with ρ2 cDNA resulted in GABA-gated whole-cell currents that differed from those mediated by the ρ1 subunit in two respects: maximal amplitude (ρ1:ρ2 ≈ 4:1) and inactivation time course (ρ1:ρ2 ≈ 2:1). Cotransfection of ρ1 and ρ2 cDNA in a 1:1 ratio generated whole-cell currents with large amplitudes characteristic of ρ1 but more rapid inactivation typical for ρ2. This observation suggested formation of heterooligomeric GABAC receptors with distinct features. Therefore, we tested the assembly of ρ1 and ρ2 subunits by cotransfecting ρ2 cDNA together with a chimeric ρ1β1 subunit, known to interfere with ρ1 assembly in a dominant-negative fashion. Reduction of ρ2 generated currents correlated with the ratio of chimeric to ρ2 cDNA. Secondly, we determined that the picrotoxinin sensitivity of cells transfected with various ratios of ρ1 and ρ2 cDNA differed from that expected of a pure mixture of homooligomeric receptors. The latter two observations support the idea that ρ1 and ρ2 subunits form heterooligomeric GABAC receptors in mammalian cells. Together, our results indicate that the presence of both ρ subunits enables the formation of heterooligomeric receptors with physical properties distinct from homooligomers, thus increasing the diversity of GABAC receptors in the CNS.