In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca²⁺ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP₃) is believed to release Ca²⁺ from the endoplasmic reticulum, but we have now tested whether the Ca²⁺-releasing messengers IP₃ and cyclic ADP-ribose (cADPr) can liberate Ca²⁺ from ZGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP₃ and cADPr evoke a marked decrease in the free intragranular Ca²⁺ concentration. Using a novel high resolution method, we have measured changes in the Ca²⁺ concentration in the vicinity of an isolated ZG and show that IP₃ and cADPr cause rapid Ca²⁺ release from the granule, explaining the agonist-evoked cytosolic Ca²⁺ rise in the secretory pole.