In pancreatic β-cells, the role of endoplasmic reticulum (ER) in the regulation of cytosolic Ca2+ concentration ([Ca2+] c) is not thoroughly understood. Moreover, the dynamics and functional role of mitochondrial Ca2+ ([Ca2+] m) as well as the interactions between ER, mitochondrial and cytosolic Ca2+ have largely been unknown. We developed a method of measuring [Ca2+] m in the insulin-secreting MIN6 cells using recombinant aequorin targeted to mitochondria, while monitoring [Ca2+] c by fura-2. In rat β-cells, glucose induced an initial decrease in [Ca2+] c, followed by an increase in [Ca2+] c due to inhibition of KATP channels and Ca2+ influx. The decrease in [Ca2+] c was due to activation of the thapsigargin-sensitive ER Ca2+-pump and Ca2+ sequestration by ER. The glucose-accelerated Ca2+ sequestration was coupled to an enhanced Ca2+ release from ER by ACh. cAMP-PKA pathway, when [Ca2+] c was elevated, also stimulated Ca2+ sequestration due to activation of ER Ca2+-pumps. In aequorin-expressing MIN6 cells, ATP induced transient increases in [Ca2+] c and [Ca2+] m. KCl (22 mM) induced a sustained increase in [Ca2+] c but a transient increase in [Ca 2+] m. The peak [Ca 2+] m response reached 1-3 μM, while that of the averaged [Ca2+] c was 300-800 nM. Therefore, microdomains with very high Ca2+ may exist in cytosol and be responsible for the transport of Ca2+ into mitochondria. Glucose induced oscillations of [Ca2+] c and, concomitantly, oscillatory and/or sustained increases in [Ca2+] m most likely result in the continuous activation of TCA cycle enzymes and ATP production, thereby leading to sustained secretion of insulin.