In vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) assemble in an unknown stoichiometry from two homologous subunits, an α and a non-α. How large is the repertoire of these subunits and how many subtypes of functionally different nAChRs can they constitute? We found in the avian genome a cluster of three closely linked genes spanning 28 kilobase pairs and encoding three proteins, nα3, α3, and α5, that have the features expected of neuronal nAChR subunits. Gene nα3 lies 5' of α3 (whose role in cholinoception has already been established) and is transcribed from the same DNA strand, whereas α5 lies 3' of α3 and is transcribed from the opposite DNA strand. The structure of the nα3 and α5 genes consists of six exons with precisely conserved splice sites and is identical to the structure of the previously characterized avian neuronal receptor subunit genes α2, α3, α4, and nα1. α3, nα3, and α5 transcripts are rare in the central nervous system, but α3 and nα3 are readily detectable in embryonic superior cervical and ciliary ganglia. In order to assay function, the gene encoding nα3 and the cDNAs encoding α3, α4, α5, and nα1 were subcloned into an expression vector, and the constructs were injected into Xenopus oocyte nuclei, either singly or in pairwise combinations of one α and one non-α. One to five days later, ACh sensitivity of the injected oocytes was examined in voltage clamp. The nα3 gene and nα1 cDNA elicited assembly of nAChRs when coinjected with α3 or α4 cDNA and the electrophysiological properties of the four pairwise combinations were significantly different. α5, however, did not direct the assembly of functional nAChRs when injected alone or in combination with nαl or nα3.