We studied the steady state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross-section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 nm and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for one- and two-photon excitation. The anisotropy decay measurements of bis-benzimide, 2,5'-bi-1H-benzimidazole, 2'-(4- ethoxphenyl)-5-(4-methyl-1-piperazinyl) (HOECHST 33342) in ethanol revealed the same correlation times for two-photon excitation as observed for one-photon excitation. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for two-photon excitation than for one-photon excitation. The anisotropy spectra of Hoechst 33342 was examined in glycerol at -20 degree(s)C, revealing limiting values close to the theoretical limits for one-photon (0.4) and two-photon (0.57) excitation. The steady-state anisotropy for one-photon excitation decreases in the shorter wavelength region (R6G dye laser, 280 to 315 nm), but the two-photon anisotropy for 560 to 630 nm excitation remains as high as in the long- wavelength region (690 to 770 nm). This result suggests that one- photon absorption is due to two electronic transitions, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm.