There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(2+)) and semi-reduced (MB) radicals of the sensitizer and subsequently super-oxide radical (O(2)(-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(-), indicating that both (1)O(2) and MB(2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).