GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABA A receptor/pore complex is presumed to be a pentamer typically composed of a combination of ␣, , and ␥ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABA A receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat ␣1(␣L263S), 2(␣L259S), and ␥2(␣L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., ␣ and ␣L263S), along with their wild-type counterparts (e.g.,  and ␥), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (␣, , and ␥) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant ␣12␥2 GABA A receptor is a pentamer composed of two ␣ subunits, two  subunits, and one ␥ subunit.