In the insulin-secreting β-cell line (STC3, stimulation with 11.2 mmol/1 glucose caused a rise in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7–36)amide and at 10⁻¹¹ mol/1; 84% of the cells responded to glucose with a rise in [Ca²⁺]_i. GLP-I(7–36)amide induces a rapid increase in [Ca²⁺]_i only in cells exposed to elevated glucose concentrations (≥5.6 mmol/1). The action of GLP-I(7–36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca²⁺ through voltage-dependent L-type Ca²⁺ channels, which then produced a further increase in [Ca²⁺]_i by mobilization from intracellular stores. The latter effect reflected Ca²⁺-induced Ca²⁺ release and was blocked by ryanodine. Similar increases in [Ca²⁺]_i were also observed in voltageclamped cells, although there was neither activation of a background (Ca²⁺-permeable) inward current nor enhancement of the voltage-dependent L-type Ca²⁺ current. These observations are consistent with GLP-I(7–36) amide inducing glucose sensitivity by promoting mobilization of Ca²⁺ from intracellular stores. We propose that this novel action of GLP-I(7–36)amide represents an important factor contributing to its insulinotropic action.