Acetylcholine is a key neurotransmitter of the inner ear efferent system. In this study, we identify two novel nAChR subunits in the inner ear: alpha1 and gamma, encoded by Chrna1 and Chrng, respectively. In situ hybridization shows that the messages of these two subunits are present in vestibular and cochlear hair cells during early development. Chrna1 and Chrng expression begin at embryonic stage E13.5 in the vestibular system and E17.5 in the organ of Corti. Chrna1 message continues through P7, whereas Chrng is undetectable at post-natal stage P6. The alpha1 and gamma subunits are known as muscle-type nAChR subunits and are surprisingly expressed in hair cells which are sensory-neural cells. We also show that ATOH1/MATH1, a transcription factor essential for hair cell development, directly activates CHRNA1 transcription. Electrophoretic mobility-shift assays and supershift assays showed that ATOH1/E47 heterodimers selectively bind on two E boxes located in the proximal promoter of CHRNA1. Thus, Chrna1 could be the first transcriptional target of ATOH1 in the inner ear. Co-expression in Xenopus oocytes of the alpha1 subunit does not change the electrophysiological properties of the alpha9alpha10 receptor. We suggest that hair cells transiently express alpha1gamma-containing nAChRs in addition to alpha9alpha10, and that these may have a role during development of the inner ear innervation.