Stimulation of pancreatic acinar cells raises [Ca2+]i via Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores, generally considered to reside within the endoplasmic reticulum (ER). However, with physiological doses of cholinergic agonists, the [Ca2+]i increase is localized to the apical (secretory) pole of the cell, leading to suggestions that zymogen (secretory) granules themselves may constitute an InsP3-sensitive Ca2+ store responsible for localized Ca2+ release. We have therefore re-investigated whether the ER in pancreatic acinar cells is capable of acting as a functional Ca2+ store in all, or only some, cellular regions. In streptolysin O-permeabilized cells, the ER accumulated up to 25 mmol of 45Ca2+ per liter ER volume by an ATP-dependent, thapsigargin-sensitive, process. This tracer Ca2+ uptake was dependent on ambient (loading) [Ca2+], as was the intra-ER free [Ca2+], assessed by imaging the fluorescence of Magfura-2 within the Ca2+ stores. Comparison of free and total intra-ER [Ca2+] indicated that 200-300 Ca2+ ions are bound within the ER lumen for every Ca2+ ion remaining free. Subcellular analysis showed that ER stores in all regions of the permeabilized cell took up Ca2+ at loading [Ca2+] between 60 nM and 1 microM. Thapsigargin released Ca2+ from stores in all cellular regions, as did InsP3. Immunofluorescence with antibodies against sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase or calreticulin confirmed that ER Ca2+ stores were present throughout the cytoplasm. In summary, these results clearly show that the endoplasmic reticulum can act as a functional Ca2+ store in all regions of the acinar cell, including the apical pole.