The genes encoding the nicotinic acetylcholine receptor alpha3, alpha5, and beta4 subunits are genomically clustered. These genes are co-expressed in a variety of cells in the peripheral and central nervous systems. Their gene products assemble in a number of stoichiometries to generate several nicotinic receptor subtypes that have distinct pharmacological and physiological properties. Signaling through these receptors is critical for a variety of fundamental biological processes. Despite their importance, the transcriptional mechanisms underlying their coordinated expression remain to be completely elucidated. By using a bioinformatics approach, we identified a highly conserved intronic sequence within the fifth intron of the alpha3 subunit gene. Reporter gene analysis demonstrated that this sequence, termed "alpha3 intron 5," inhibits the transcriptional activities of the alpha3 and beta4 subunit gene promoters. This repressive activity is position- and orientation-independent. Importantly, repression occurs in a cell type-specific manner, being present in cells that do not express the receptor genes or expresses them at very low levels. Electrophoretic mobility shift assays demonstrate that nuclear proteins specifically interact with alpha3 intron 5 at two distinct sites. We propose that this intronic repressor element is important for the restricted expression patterns of the nicotinic receptor alpha3 and beta4 subunit genes.