The regulation of organelle free Ca2+ was analysed in individual mouse pancreatic beta-cells loaded with the fluorescent low-affinity indicator furaptra. Removal of the cytoplasmic indicator by controlled digitonin permeabilization of the plasma membrane resulted in a sudden increase of the 340 nm/380 nm fluorescence excitation ratio followed by a gradual decay, reflecting the emptying of Ca2+ from organelle pools. Subsequent introduction of 3 mM ATP caused rapid refilling of a Ca2+ pool, which represented the endoplasmic reticulum (ER) in being mobilized with inositol 1,4,5-trisphosphate (IP3) and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. The concentration of Ca2+ in the ER observed immediately after permeabilization depended on the glucose concentration in a hyperbolic fashion with half-maximal filling at about 6 mM of the sugar. Glucose promotion of Ca2+ sequestration in the ER involved a high-affinity mechanism not requiring but accelerated by a rise of the cytoplasmic Ca2+ concentration. Glucose also exerted a long-term action on the ER storage of Ca2+, maintaining the set-point for its maximal concentration and preserving the response to IP3. The results indicate that the ER has an important role in the glucose-stimulated beta-cell by serving as a high-affinity sink for Ca2+, irrespective of the prevailing concentration of cytoplasmic Ca2+.