The store-operated pathway for Ca(2+) entry was studied in individual mouse pancreatic beta-cells by measuring the cytoplasmic concentrations of Ca(2+) ([Ca(2+)](i)) and Mn(2+) ([Mn(2+)](i)) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca(2+) sequestration. To avoid interference with voltage-dependent Ca(2+) entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca(2+) depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca(2+)](i) or from the rate of increase in [Mn(2+)](i) due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn(2+) depended on store filling in a graded manner. The store-operated influx of Ca(2+)/Mn(2+) was inhibited by Gd(3+) and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the beta-cell has direct implications for the control of membrane potential and insulin secretion.