Inositol trisphosphate (IP₃) and cyclic ADP-ribose (cADPR) release Ca²⁺ from the endoplasmic reticulum (ER) via IP₃ and ryanodine receptors (RyRs), respectively. In contrast, nicotinic acid adenine dinucleotide phosphate (NAADP) may activate a novel Ca²⁺ channel in an acid compartment. We have used two photon permeabilized pancreatic acinar cells. Cells were isolated from CD1 male nouse pancreas (animals were humanely killed). We have shown that the three messengers tested could each release Ca²⁺ from the ER (n=25, 16 and 30, respectively) and also from an acid store in the granular region (n=10, 8 and 20, respectively). The NAADP action on both types of store, like that of cADPR but unlike IP₃, depended on operational RyRs, since it was blocked by high concentration of ryanodine (100 μM) or ruthenium red (10 μM) (n=5 and 9, respectively). In the whole cell the acidic store is approximately half of the size of the ER (47±11%, S.D., n=7). In the secretory granular area, the acidic store is 30±5% larger than the ER Ca²⁺ store (n=8). We estimate that the free [Ca²⁺] in the acidic store is 300±70 μM, whereas the free [Ca²⁺] in the ER is 120±50 μM (n=3). The acid Ca²⁺ store in the granular region did not have Golgi or lysosomal characteristics and is therefore most likely in the secretory granules. The ER is predominantly basal, but thin extensions penetrate into the granular area and Ca²⁺ signals probably initiate at sites where ER elements and granules come close together.