How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles (“organelle selection”) to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively select the ER Ca2+ store, whereas cholecystokinin additionally recruits a lysosome-related organelle. Similarly, in a pancreatic β cell line MIN6, acetylcholine selects only the ER, whereas glucose mobilizes Ca2+ from a lysosome-related organelle. We also show that the key to organelle selection is the agonist-specific coupling messenger(s) such that the ER is selected by recruitment of inositol 1,4,5-trisphosphate (or cADP-ribose), whereas lysosome-related organelles are selected by NAADP.