Efferents, originating in the superior olivary complex, preferentially synapse with cochlear outer hair cells (OHCs), with acetylcholine (ACh) as their primary neurotransmitter. The OHC ACh receptors (AChRs), which have unusual pharmacology, have been cloned and identified as a new subunit (alpha9) of the nicotinic AChR family. The expression of alpha9 AChRs is first detected before birth and peaks between 6 and 10 days after birth (DAB) in developing mice and rats, while functional maturation of the receptor, as determined by measuring the ACh-induced currents, takes place between 6 and 12 DAB. In this study we attempted to examine the development of AChRs in OHCs grown in explanted cultures, deprived of efferent innervation. ACh-induced currents were used as an assay. Reverse transcription-PCR analysis was also performed to detect the expression of alpha9 subunit from cultured OHCs. PCR study indicates that mRNA of the alpha9 subunit was expressed in primary cochlear cultures, similar to that seen in the cochleae of developing animals. Measurement of whole-cell currents showed that ACh-induced outward current was first detected around 5 days in a fraction of cultured OHCs. The number of responsive cells increased between 5 and 12 days in culture. The size of ACh-induced currents also increased during this period. These results suggest that the development of AChRs in cultured OHCs is not affected by removal of efferent innervation.