We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 subunit a reporter mutation (V273T), which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.