We report emission spectra and time-resolved intensity and anisotropy decays resulting from three-photon excitation of fluorescence. The emission intensity of 2,5-diphenyloxazole (PPO) was found to depend on the third power of the excitation intensity at 870 nm. This wavelength is too long to excite PPO by a two-photon process. The emission spectrum, intensity decay, and rotational correlation times were found to be identical for one- (290 nm) and three-photon (870 nm) excitation. However, the time-zero anisotropy (r0 = 0.61) observed from the time-resolved anisotropy decay, and observed in frozen solution, was larger than for one- or two-photon excitation. The possibility of three-photon excitation with a Ti:sapphire laser suggests wide ranging applications in biophysics and fluorescence microscopy.