The insulin secretagogue D-glucose induces both accumulation of nonesterified arachidonic acid (35 μM) in pancreatic islets and a rise in beta cell cytosolic [Ca⁺⁺]_i. Arachidonate amplifies both voltage-dependent Ca⁺⁺ entry in secretory cells and depolarization-induced insulin secretion. Here, arachidonate induced a biphasic rise in [Ca⁺⁺]_i of Fura-2AM loaded beta cells which increased with arachidonate concentration (5–30 μM), was reversed upon washout, and was unaffected by the arachidonate oxygenase inhibitor BW755C. The sustained phase of the rise was abolished by removal of extracellular Ca⁺⁺ and amplified by depolarization with KCl. The accumulation of nonesterified arachidonate in islets stimulated by D-glucose may therefore promote the D-glucose-induced rise in beta cell [Ca⁺⁺]_i.