One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different beta subunits assemble with an alpha subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for alpha3, beta2, and beta4 subunits. Beta2 and beta4, in pairwise combination with alpha3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). Alpha3beta4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through alpha3beta2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for alpha3, beta2, and beta4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both beta2 and beta4 subunits in functional receptors.