To minimize the effects of Ca²⁺ buffering and signaling, this study sought to examine single Ca²⁺ channel properties using Sr²⁺ ions, which substitute well for Ca²⁺, but binds weakly to intracellular Ca²⁺ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity towards dihydropyridine antagonist (non L-type). The L- and non L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr²⁺ and 11 and 13 pS at 5 mM Sr²⁺, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr²⁺ for L- and non L-type channels, respectively. At Ca²⁺ concentration of ~2 mM, the single-channel conductances of Sr²⁺ for the L-type channel was ~1.5 pS and 4.0 pS for the non L-type channels. Thus, the limits of single-channel microdomain at the membrane potential of a hair cell (e.g. -65 mV) for Sr2+ ranges from 800-2000 ion ms-1, assuming a ECa of 100 mV. The channels are at least 4-fold more sensitive at the physiological concentration ranges than at concentrations > 10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr²⁺, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca²⁺ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr²⁺ may represent divalent ion-dependent inactivation of the L-type channel.