We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole hydrochloride) and Hoechst 33342 (bis- benzimide, or 2,5'-bi-1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1- piperazinyl)) with two-photon (2hv) and three-photon (3hv) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three- photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emissior spectra and decay times for 2hv and 3hv excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3hv at 885 nm than for 2hv at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.