Each of these channel types plays an essential role in hearing, and all have shown sensitivity to membrane cholesterol content in various cellular models. The large conductance, calcium-activated, ‘BK’-type potassium channels play a variety of roles in the physiological functions of numerous cellular systems. In inner ear hair cells, BK channels are responsible for the temporal precision of sound encoding in mammals. In non-mammals, BK expression and kinetics are responsible for setting the resonant frequency of afferentlyinnervated hair cells. BK channels shape the receptor potential generated by inner hair cells, as evidenced by slowed voltage responses of inner hair cells in mice lacking the pore-forming alpha subunit of BK. The importance of BK channels in audition is underpinned by their expression at the onset of hearing. While cholesterol reportedly modulates BK currents in smooth muscle, glioma, neuronal and endothelial cells, a functional role in auditory hair cells has not been reported previously. In non-mammals, L-type voltage-gated calcium channels serve as the calcium source for BK channels. In all vertebrates, L-type VGCCs also play a key role in neural transmission from the hair cell to the auditory nerve, triggering exocytosis at the basolateral end of the hair cell. Due to high calcium buffering, VGCCs must be in close proximity to BK and to the calcium sensors driving vesicular fusion. Cholesterol inhibits L-type VGCCs in cardiac and coronary myocytes, where cholesterol chelation with MbCD enhances channel activity. However, similarly to the BK channel, the role of cholesterol in Vorinostat modulating VGCCs varies depending upon the particular preparation and cell type studied. Kv and Kir channels counteract depolarization and hyperpolarization of the cell membrane respectively. Kv and Kir channels underlie tuning in the low frequency, apical hair cells of nonmammals. Kv currents play a specialized role in hair cell development, repolarizing the spontaneous action potentials credited with directing the tonotopic organization of the auditory periphery. Cholesterol depletion with MbCD potentiates Kv currents in developing auditory hair cells and abolishes SAPs. Kir currents display sensitivity to cholesterol depletion in a variety of cell types, but the role of cholesterol in modulating Kir in auditory hair cells is unknown. We investigated the effects of the cholesterol depleting drug, MbCD, on the macroscopic currents of the four major ion channel classes responsible for determining the membrane potential of chick auditory hair cells. Depleting cholesterol from the hair cell membrane reduced BK currents while VGCC conductance was increased. There were lesser or absent effects of cholesterol depletion on Kv and Kir currents respectively. Cholesterol staining was most intense at the apical and basolateral ends of the cell and BK channels were identified in cholesterol-enriched microdomains. Our data show that the lipid environment modulates ion channels essential for sound processing and synaptic transmission in the auditory hair cell membrane.