Cytosolic calcium signals are produced by suddenly increasing the concentration of free calcium ions (Ca2+). This can occur by opening channels permeable to Ca2+ either in the surface cell membrane or in the membranes of intracellular organelles containing high Ca2+ concentrations. Ca2+ signals can control several different processes, even in the same cell. In pancreatic acinar cells, for example, Ca2+ signals do not only control the normal secretion of digestive enzymes, but can also activate autodigestion and programmed cell death. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited. The mechanisms responsible for Ca2+ signal compartmentalization are now largely known and will be described on the basis of recent studies of Ca2+ transport pathways and their regulation in pancreatic acinar cells. It turns out that the Ca2+ handling as well as the structural characteristics of the endoplasmic reticulum (ER) and the mitochondria are of particular importance. Using a variety of Ca2+-sensitive fluorescent probes placed in different sub-cellular compartments in combination with local uncaging of caged Ca2+, many new insights into Ca2+ signal generation, compartmentalization and termination have recently been obtained