Heteromeric glycine receptors mediate synaptic inhibition in the caudal areas of the adult mammalian central nervous system (CNS). These channels resemble other receptors in the nicotinic superfamily in that they are pentamers, but may differ in that they contain alpha and beta subunits in a 3:2 rather than a 2:3 ratio. Evidence in favor of a 3alpha:2beta stoichiometry of heteromeric glycine receptors comes from biochemical data and from the expression of chimeric subunits. We investigated this question using a potentially more direct approach and mutated the highly conserved hydrophobic residues in the middle (position 9') of the pore-lining domain. This mutation increases agonist potency in all channels in the nicotinic superfamily and its effects are in first approximation proportional to the number of mutant subunit incorporated into the receptor. We expressed in HEK 293 cells wild-type glycine alpha1beta receptors or receptors bearing the 9' mutation on either the alpha or the beta subunit, using an alpha:beta plasmid ratio of 1:40 in the transfection. This resulted in negligible levels of contamination by homomeric alpha1 receptors, as proven by low picrotoxin potency and by the extreme rarity of high conductances in single channel recording. Our data show that the effects of the 9' mutation on the receptor sensitivity to glycine were more marked when the alpha subunit bore the mutation. The magnitude of the leftward shift in the agonist dose-response curve for the two mutant combinations was in agreement with a subunit stoichiometry of 3alpha:2beta.