Abstract We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 μM Ca 2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.