Previous results demonstrated that nimodipine, an L-type of Ca2+ channel antagonist, abolished the negative summating potential (SP) recorded from anesthetized guinea pigs (Bobbin et al., 1990), suggesting that Ca2+ is involved in generation of the negative SP. Therefore we examined the effect of changing concentrations of perilymph Ca2+ on this cochlear potential. Perilymph spaces of guinea pig cochleae were perfused with artificial perilymph solutions containing zero mM Ca2+, zero mM Ca2+ with 2 mM EGTA, 30 mM Mg2+ and increasing levels of Ca2+ (2, 4, 8, 16 mM) at a rate of 2.5 microliters/min for 10 min. Immediately after each period of perfusion the compound action potential of the auditory nerve (CAP), cochlear microphonics (CM) and the negative SP evoked by 10 kHz tone bursts of varying intensities were recorded from a wire inserted in the basal turn scala vestibuli. Decreasing the level of Ca2+ decreased the magnitude of the negative SP, whereas increasing the level of Ca2+ progressively increased the magnitude of the negative SP. Mg2+ (30 mM) suppressed the CAP to the same extent as zero mM Ca2+ with 2 mM EGTA, but only slightly increased the magnitude of the negative SP. These results support the hypothesis that Ca2+ and L-type Ca2+ channels are involved in the function of the hair cells and the generation of the negative SP. Mg2+ appears to be a selective antagonist of the Ca2+ channel involved in transmitter release.