It is known that the stimulation with high glucose initially decreases as well as subsequently increases the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. In the present study, we aimed at exploring the ionic mechanism and physiological role of the glucose-induced decrease in [Ca2+]i by measuring [Ca2+]i in single pancreatic beta-cells from normal rats. The glucose-induced decrease in [Ca2+]i in beta-cells was completely inhibited by thapsigargin (Tg), a specific inhibitor of the endoplasmic reticulum (ER) Ca2+ pump (Ca(2+)-ATPase). On the other hand, neither a Ca(2+)-free nor a low-Na+ condition significantly altered the glucose-induced decrease in [Ca2+]i. At basal glucose concentrations (1-4.5 mM), an insulin secretagogue acetylcholine (ACh) evoked a rather transient increase in [Ca2+]i in the presence and absence of extracellular Ca2+. A rise in glucose concentration from 1 to 4.5 mM produced a sustained decrease in [Ca2+]i and concomitantly augmented the ACh-evoked increase in [Ca2+]i. The resting [Ca2+]i level determined by glucose was tightly and reciprocally correlated with the peak of the [Ca2+]i response to ACh. Successive ACh pulses elicited repeated [Ca2+]i responses, which were progressively inhibited by Tg, suggesting that Ca2+ released by ACh was taken up by the ER Ca2+ pump and thus cycled. The results demonstrate that glucose decreases [Ca2+]i in pancreatic beta-cells mainly by activating the Ca2+ pump in ER from which ACh mobilizes Ca2+. Furthermore, the glucose-stimulated sequestration of Ca2+ by ER results in an augmented [Ca2+]i response to ACh, providing a mechanistic basis for the glucose-dependent action of ACh to initiate insulin secretion.