Using pancreatic beta-cells isolated from obese hyperglycemic mice, it was demonstrated that the addition of 5 mM extracellular Ca2+ evoked a rapid and transient increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). The effect remained in the presence of D-600. Extracellular Ca2+ did not raise [Ca2+]i subsequent to emptying the inositol 1,4,5-trisphosphate (InsP3) sensitive pool by carbamylcholine stimulation, indicating that the pool released by extracellular Ca2+ is of similar origin. Stimulation with extracellular Ca2+ was accompanied by a pronounced insulin release. Our results suggest that the Ca2+-induced rise in [Ca2+]i is mediated through the formation of InsP3, a mechanism that might operate also in other types of cells.