Intracellular Ca
2+ acts as the second messenger in a variety of cells. Many cellular functions are tightly regulated by the intracellular Ca
2+ concentrations ([Ca
2+]
i). Therefore, measurement of the [Ca
2+]
i is of critical importance. Calcium-sensitive dual excitations, indicators, such as fura-2, are now widely used to measure the [Ca
2+]
i in living cells. The development of techniques allowing the measurement of [Ca
2+]
i has contributed noticeably to our understanding of many cellular functions. Digital video microscopy, confocal laser scanning microscopy, and multiphoton microscopy allow accurate spatial analysis of [Ca
2+]
i at the subcellular level. Fura-2 has been used commonly to measure the [Ca
2+]
i because of the sensitivity and specificity of the method. It can be loaded into living cells with little disruption of the cellular functions. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used extracellularly because of its ability to pass through cell membranes, whereas fura-2 by itself cannot be introduced into the cells. For fura-2 measurements, measurements of the fluorescence intensities at two excitation wavelengths can be used to obtain an estimate of the [Ca
2+]
i, independent of the dye concentration and the thickness of the cell membrane. In this review, I summarize the advantages and pitfalls of using fura-2 and other standard Ca
2+ indicators, methods used to measure the [Ca
2+]
i, including simultaneous measurements of cell movements and the changes in the [Ca
2+]
i, and procedures used for measuring the cellular and/or subcellular Ca
2+ concentrations in living cells from the cochlea, such as the outer and inner hair cells.
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