Mechanically gated "transduction" channels in inner ear hair cells are thought to be connected to tip links stretched between adjacent stereocilia. To locate active channels, calcium-green fluorescence in single stereocilia was measured with two-photon laser scanning microscopy. Bundle deflection increased fluorescence in many but not all stereocilia; the increase was blocked by depolarization. The number of stereocilia responding was proportional to the transduction current, consistent with Ca2+ influx through transduction channels. Fluorescence rose first in the tips of stereocilia and then in the bases, in agreement with channel localization at the tips. Some of the shortest stereocilia in a bundle showed a fluorescence increase, as did some of the tallest, indicating that transduction channels can be at either or both ends of tip links.