Picrotoxin, a potent antagonist of the inhibitory central nervous system GABAA and glycine receptors, is believed to interact with residues that line the central ion pore. These pore-lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6' location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the alpha, beta or gamma subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two alpha subunits, two beta subunits and one gamma subunit, it is not clear how many alpha and beta subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant alpha or beta subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6' mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.