GABAA receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of alpha, beta, and gamma subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant alpha1beta3gamma2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABAA receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two alpha subunits, two beta subunits, and one gamma subunit was derived. Similar experiments investigating the composition of alpha1beta3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with alpha1 and beta3 subunits resulted in a stoichiometry of two alpha and three beta subunits. Density gradient centrifugation studies indicated that combinations of alpha1beta3gamma2 or alpha1beta3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of alpha1gamma2 or beta3gamma2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABAA receptor assembly and support the conclusion that GABAA receptors are pentamers in which a total of four alternating alpha and beta subunits are connected by a gamma subunit.