Ligand-gated ion channels are multi-subunit complexes where each subunit-type is encoded by several related genes. Heterologous expression of any one of the neuronal nicotinic acetylcholine receptors (nAChR) alpha-type subunits, either alone or with any beta-type subunit, typically yields functional nAChR channels. A striking exception is the nAChR alpha5 subunit: although apparently complexed with beta2 and beta4 nAChR subunits in neurons, and expressed in a subset of neurons within the central and peripheral nervous systems, heterologous expression of alpha5, either alone or with any beta-type subunit has failed to yield functional channels. We demonstrate here that alpha5 does participate in nAChRs expressed in hetrologous systems and in primary neurons, and further that alpha5 contributes to the lining of functionally unique nAChR channels, but only if coexpressed with both another alpha- and beta-type subunit. Furthermore, channels containing the alpha5 subunit are potently activated and desensitized by nanomolar concentrations of nicotine.