We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca 2+ store or is composed of separate subunits by monitoring movements of Ca 2+ and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca 2+ and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca 2+ store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca 2+ concentration throughout the cell, but repetitive cytosolic Ca 2+ spikes, induced by a low ACh concentration, hardly reduced the ER Ca 2+ level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca 2+ liberation. Ca 2+ released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.