Outer hair cells (OHCs), the sensory-motor cells responsible for the extraordinary frequency selectivity and dynamic range of the cochlea, rapidly endocytose membrane and protein at their apical surface. Endocytosis and transcytosis in isolated OHCs from the mature guinea-pig cochlea were investigated using the amphiphatic membrane probe FM1-43. We observed membrane transport from the apical surface to both the basolateral wall and the subnuclear pole. By double-labelling with DiOC6, a stain for endoplasmic reticulum, and aspiration of the plasma membrane, we showed that the basolateral target was the subsurface cisternae. The fluorescent signal was about three times weaker at the basal than at the apical pole. The speed of vesicle transport to the subnuclear pole was approximately 0.4 μm/s. Changing extracellular Ca2+ concentration from 25 μM to 2 mM accelerated rapid endocytosis. Extracellular application of BAPTA-AM (25 μM), an intracellular Ca2+ chelator, and TFP (20 μM), a specific inhibitor of calmodulin, reduced endocytic activity, as did depolarization of the whole cell. The presence of extracellular Cd2+ (200 μM), a Ca2+-channel blocker, had no effect on the voltage dependence of endocytosis at the apical pole, and inhibited the voltage dependence at the subnuclear pole. These results suggest that rapid endocytosis is a Ca2+/calmodulin-dependent process, with extracellular Ca2+ entering through voltage-gated Ca2+ channels at the basal pole. The two distinct destinations of endocytosed membrane are consistent with the functional polarization of the OHC, with the basolateral wall being dedicated to electromechanical transduction and the subnuclear pole being dedicated to electrochemical transduction processes.