Two-photon laser flash photolysis was used to investigate the wavelength dependence of upper excited state chemistry of Rose Bengal (RE) in order to rationalize differences in RE-sensitized inhibition of acetylcholinesterase (ACE) in red blood cell ghosts, observed using different irradiation modes. The absorption of the RE triplet state, generated using 532-nm irradiation, was monitored as a function of wavelength used for subsequent excitation to upper triplet levels. Triplet state bleaching, due to formation of radical species, was observed using wavelengths greater than similar to 580 nm to excite into an upper state, T-n. However, no bleaching was observed at lower wavelengths, corresponding to higher photon energies, but an increase in emission intensity from the sample, consistent with S-1 - S-0 fluorescence, was observed following the second pulse. A mechanism for this wavelength-dependent photochemistry is provided, which considers the relative positions of upper singlet and triplet states. Reverse intersystem crossing, and hence repopulation of the triplet state, are only observed when the energy of the second photon is sufficient to access a triplet state (Tn+1) of similar energy to the S-2 state. At lower photon energies (higher wavelengths), a lower energy state (T-n) state is accessed, from which radical formation and bleaching of the triplet state ensues.