Using a reporter mutation approach and the two-electrode voltage clamp technique, Groot-Komielink et al (1998) have shown that the 'orphan' α3 subunit can be incorporated into a functional 'triplet' α3β4α3 neuronal nicotinic acetylcholine receptor, when expressed in Xenopus oocytes. Based on the observation that in α3β4 receptors the EC50 value decreases with the number of 9'T mutations, we used the same reporter mutation approach to determine the stoichiometry of 'triplet' receptors. When either α3 or β4 carried the 9'T mutation in 'triplet' α3β4α3 or α3β4α5 receptors, the observed decreases in EC50 values were similar and larger than if only α3 or α5 were mutated. This suggests α3 and β4 are present in equal copy number, greater than that of α3 or α5, namely; 2 copies of β, 2 copies of β and 1 copy of α3 or α5. Potency ratio data for α3β4 and α3β4α3 receptors demonstrated that the incorporation of α3 reduced the potency of lobeline (relative to ACh) from 23.0 to 7.14 (p 0.01). However, the rank order of the seven nicotinic agonists tested was unchanged. Schild analysis showed that the Kd for the competitive antagonist trimetaphan was not affected by α3 incorporation (75.5 and 66.0 nM for α3β4 and α3β4α3, respectively), suggesting that α3 may not contribute to the properties of the agonist binding site. Calcium permeability data failed to detect differences in calcium permeability for α3β4 and α3β4α3 receptors, with mean shifts in reversal potential of 7.68 mV for α3β4 and 5.20 mV for α3β4α3 (for a 10 times increase in external calcium concentration)