A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.