The neuronal nicotinic subunit beta3 forms functional receptors when co-expressed with both an alpha and a beta subunit, such as alpha3 and beta4. We examined the subunit stoichiometry of these 'triplet' alpha3beta4beta3 receptors by expression in Xenopus oocytes of the alpha3, beta4 and beta3 subunits, either in wild-type form or after insertion of a reporter mutation. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9THORN ) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. In alpha3beta4beta3 receptors, the ACh EC50 was decreased by approximately 17-fold when this mutation was in alpha3 alone and only by fourfold when beta3 alone was mutated. Mutating beta4 was equivalent to mutating alpha3, suggesting that the 'triplet' receptor contains one copy of beta3 and two copies each of alpha3 and beta4. Mutating beta3 and alpha3 or beta3 and beta4 reduced the ACh EC50 further, to values two- to threefold lower than those seen when only alpha3 or beta4 carried the mutation. In 'pair' alpha3beta4 receptors (known to contain two alpha and three beta subunits), mutating beta4 had a greater effect on the ACh EC50 than mutating alpha3, in agreement with an alpha:beta ratio of 2:3 and a constant and independent effect of each copy of the mutation. Our results suggest that alpha3beta4beta3 neuronal nicotinic receptors contain one copy of beta3 and two copies each of alpha3 and beta4 and confirm that in pair alpha3beta4 receptors the alpha/beta subunits are present in a 2:3 ratio.