alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3-M4 loop. A third nicotinic subunit message, termed alpha9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for alpha9-III was not detected in hair cells. Message for alpha9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, alpha9-I and alpha9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of alpha10 appears to exist. The current work is, to our knowledge, the first description of alpha9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the alpha9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.