We observed emission from the tyrosine derivative N-acetyl-l-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5°C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable. Copyright (C) 1999 Elsevier Science B.V. All rights reserved.