Electrical resonance, which in some hair cells provides a mechanism for frequency tuning, is mediated by clusters of Ca2+ channels and Ca(2+)-activated K+ channels that have been proposed to occur at presynaptic active zones. To localize Ca2+ channels on the cellular surface, we loaded hair cells from the frog's sacculus with the Ca2+ indicator fluo-3 and imaged them by fluorescence confocal microscopy. When a cell was depolarized, we observed on its basolateral surface several foci of transiently enhanced fluorescence due to local Ca2+ influx. After protracted recording, each cell displayed on average 18 brightly and permanently fluorescent spots at the same positions. We mapped these spots in four hair cells and compared their locations with those of presynaptic active zones, as determined from transmission electron micrographs of serial sections through the same cells. The results demonstrated that enhanced fluo-3 fluorescence marks active zones. Measurement of currents through membrane patches at fluorescently labeled active zones demonstrated that both voltage-activated Ca2+ channels and Ca(2+)-activated K+ channels occur there. These results confirm that the ion channels involved in electrical tuning and synaptic transmission by hair cells cluster together at presynaptic active zones.