Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.