When sound stimulates the stereocilia on the sensory cells in the hearing organ, Ca²⁺ ions flow through mechanically gated ion channels. This Ca²⁺ influx is thought to be important for ensuring that the mechanically gated channels operate within their most sensitive response region, setting the fraction of channels open at rest, and possibly for the continued maintenance of stereocilia. Since the extracellular Ca²⁺ concentration will affect the amount of Ca²⁺ entering during stimulation, it is important to determine the level of the ion close to the sensory cells. Using fluorescence imaging and fluorescence correlation spectroscopy, we measured the Ca²⁺ concentration near guinea pig stereocilia in situ. Surprisingly, we found that an acellular accessory structure close to the stereocilia, the tectorial membrane, had much higher Ca²⁺ than the surrounding fluid. Loud sounds depleted Ca²⁺ from the tectorial membrane, and Ca²⁺ manipulations had large effects on hair cell function. Hence, the tectorial membrane contributes to control of hearing sensitivity by influencing the ionic environment around the stereocilia.