Hair cells in the auditory sensory organ are specialized mechanoreceptors common to mammalian and non-mammalian species. The mammalian cochlear outer hair cells (OHC) possess a distinct motile property, dubbed membrane-based electromotility, that enhances the receptor function. This electromotility is believed to be the basis of cochlear amplification that increases sensitivity of the mammalian ear to sound. Prestin, a unique voltage-sensitive motor molecule localized in the lateral membrane of OHC, is presumably responsible for OHC electromotility. It has been documented that prestin null-animals lack electromotility and suffer from approximately 50 dB loss of hearing sensitivity. To identify proteins that interact with prestin we carried out a yeast two-hybrid library screen using the C-terminal intracellular domain of prestin as bait. Seven bait-dependent prey clones were identified independently. Further analysis revealed that they encode partially over-lapping regions of a single protein: a transcriptional repressor, promyleocytic leukemia zinc finger protein (PLZF). PLZF encodes a POZ/domain Kruppel-type zinc finger transcription factor reported to have pro-apoptotic and anti-proliferative activity. The interaction between endogenous prestin and PLZF proteins in the cochlea was confirmed by co-immunoprecipitation using organ of Corti lysates. Furthermore, immunohistochemical studies strongly suggest that PLZF co-localizes with prestin near the lateral membrane of cochlear OHC. The physiological significance of this interaction remains to be explored.