We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single- photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two- photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three- photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6θ photoselection, as compared with cos2θ photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium- dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium- dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.