Changes in cytosolic free Ca 2+ concentration ([Ca 2+ ] c ) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca 2+ influx (mainly through voltage-dependent Ca 2+ channels, but also through voltage-independent Ca 2+ channels like store-operated channels) and efflux pathways. Changes in [Ca 2+ ] c directly affect [Ca 2+ ] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca 2+ influx and efflux pathways, they mutually influence free [Ca 2+ ] in the others. In this article, we review the mechanisms of control of [Ca 2+ ] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca 2+ ] ER ), acidic stores and mitochondrial matrix ([Ca 2+ ] mito ), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca 2+ homeostasis in Type 2 diabetes.