We report the first measurements of protein fluorescence with three- photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three- photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1L(b) state of tryptophan from 830 to 900 nm.