The spatial characteristics of inositol 1,4,5-trisphosphate (IP3)-induced quantal Ca2+ release were examined by imaging Ca2+ concentrations within Ca2+ stores ([Ca2+]L) in permeabilized HSY cells. The image of mag-fura-2 fluorescent ratio with dual excitation (344 nm/360 nm) demonstrated that a sequential application of different concentrations of IP3 (0.1, 0.3, 10 microM) resulted in a stepwise decrease in the ratio at all regions of the cytoplasm. This change in the ratio suggests that the stepwise decrease in [Ca2+]L is associated with the quantal Ca2+ release. To monitor the change in [Ca2+]L within a single organelle, IP3-dependent changes in the mag-fura-red fluorescence of permeabilized cells were studied by confocal microscopy. Applications of increasing concentrations of IP3 caused a stepwise increase in fluorescence within ER-like reticulum structures of the cytoplasm. This finding suggests that the [Ca2+]L in a single Ca2+ store was not depleted by submaximal concentrations of IP3, and supports the steady-state model of quantal Ca2+ release.