Isolated cochlear outer hair cells undergo rapid, force-generating length changes in response to electrical stimulation. The cellular mechanism responsible for electromotility and its structural substrate is not yet known. Salicylates reduce and block electromotility in vitro. Therefore, we exposed isolated outer hair cells from the guinea pig cochlea to various doses of sodium salicylate and evaluated both ultrastructural changes and responses to electrical stimulation. Following salicylate superfusion, the subsurface cisternae showed dilatation, vesiculation and a deviation from their normal, unfenestrated, axial orientation below the plasma membrane. These changes were time and dose dependent and reversible over a time course of about 30 min. Electromotility was blocked and showed recovery following the same time course as the salicylate-induced reversible structural changes. These results indicate that intact, unfenestrated subsurface cisternae are required for the optimal generation of electrically-induced motility in mammalian outer hair cells.