Where cholesterol depletion augments VGCCs and inhibits BK channels, with absent or less robust effects on Kir and Kv channels respectively. This observation argues against a unifying, global mechanism of MbCD influence on channel function and favors more restricted effects such as disrupting direct cholesterol-channel binding and/ or producing localized effects on a heterogeneous lipid environment. Impacts of cholesterol on channel biophysics are supported by significant effects of MbCD on maximal conductance, and the change in IK,fast slope implies a direct effect of MbCD on BK channel number or gating. Disruption of cholesterol-enriched microdomainsmay also contribute to the MbCD effects. As a calcium-activated channel, BK requires close proximity to its calcium source to be efficiently activated upon depolarization, and modeling suggests that calcium-dependent BK channel activation requires VGCCs to be within tens of nanometers of the channel in order to overcome intracellular buffering. The present immunoblotting data and filipin staining are consistent with the localization of BK channels to cholesterol-enriched microdomains at the hair cell base. The IK,fast I-V curve shows a,10 mV rightward shift which may be due to either a direct effect on gating or displacement of BK channels from the VGCC calcium source, as decreased intracellular calcium concentration produces a rightward shift in activation. It is possible that the increase in calcium conductance following MbCD treatment partially compensates for displacement of VGCCs and BK channels from their normal co-clustered arrangement at the hair cell base. Membrane cholesterol in the hair cell may have specialized roles at the apical and basal ends of this complex sensory receptor. Our results reveal a new role for cholesterol in the regulation of VGCCs and BK channels which are clustered at the base of mature auditory hair cells. The functional significance of cholesterol at the hair cell apex is unknown, but our observations of ‘floppy’ hair bundles following MbCD treatment indicate a role in the structural stabilization of the rootlet. The rootlet is a filamentous structure that anchors each mechanosensitivestereocilium into the cuticular plate. The details of how and why cholesterol performs this structural role are open to speculation, but the clear implications for mechanotransduction warrant further investigation. The role of the lipid environment in auditory hair cell physiology is only beginning to be explored. In addition to reported roles in OHC function and delayed rectifier development, our results show that cholesterol influences the VGCC and BK channels necessary for sound encoding. Cholesterol exerts its influence through direct biophysical effects on these channels and may affect the interplay between them via a clustering mechanism. A potential link between the lipid environment and auditory mechanotransduction is a virtual unknown and should be explored. Cholesterol modulation of BK and VGCC channel conductances in hair cells expand our understanding of the mechanisms MG132 influencing auditory hair cell excitability and may provide novel pathways for therapies intervening in sensorineural hearing loss.