The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.