Evidence has accrued during the past two decades that mitochondrial Ca2+ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca2+ transport systems might be able to relay the changes of cytosolic Ca2+ concentration ([Ca2+]c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca2+ concentration ([Ca2+]m) become essential. In this article, we review the results from recent studies attempting to measure [Ca2+]m in living cells. In addition, the significance of each study is discussed.