K_ATP channel activity influences beta cell Ca²⁺ homeostasis by regulating Ca²⁺ influx through L-type Ca²⁺ channels. The present paper demonstrates that loss of K_ATP channel activity due to pharmacologic or genetic ablation affects Ca²⁺ storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca²⁺ release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca²⁺ ATPases by cyclopiazonic acid abolished the FCCP-induced Ca²⁺ transient. In beta cells treated with K_ATP channel inhibitors FCCP elicited a significantly larger Ca²⁺ transient. Cyclopiazonic acid did not abolish this Ca²⁺ transient suggesting that non-ER compartments are recruited as additional Ca²⁺ stores in beta cells lacking K_ATP channel activity. Genetic ablation of K_ATP channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca²⁺-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca²⁺ evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K_ATP channel activity conveys Ca²⁺ release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca²⁺ it is suggested that mitochondria are the recruited store. The change in Ca²⁺ sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.