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The oxidation of the fluorescent dye 2,7-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF ⅐ ). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF ⅐ oxidizes GSH, generating the glutathione thiyl radical (GS ⅐ ), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS ⅐ radical. Similarly, DCF ⅐ oxidized NADH, generating the NAD ⅐ radical that reduced oxygen to superoxide (O 2 . ), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H 2 O 2 , which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H 2 O 2 production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.2Ј,7Ј-Dichlorofluorescin (DCFH) 1 is widely used to measure oxidative stress in cells. The diacetate form of DCFH enters the cell and is hydrolyzed by intracellular esterases to liberate DCFH. Upon reaction with oxidizing species, the highly fluorescent compound 2Ј,7Ј-dichlorofluorescein (DCF) is formed. The fluorescence intensity can be measured and is the basis of the popular cellular assay for oxidative stress (1).Although this assay is commonly used, there are many controversies regarding its real validity. It is not clear which oxidative species is responsible for the oxidation of DCFH. Furthermore, there are some disagreements in the literature concerning the effect of superoxide dismutase on oxidative stress in cells monitored by the DCF fluorometric assay. Some studies report an inhibitory effect by superoxide dismutase (2-15), while almost the same number of studies report no effect (16 -26). A recent paper by LeBel et al. (27) mentioned that the interpretation of specific reactive oxygen species involved in the oxidation of DCFH to DCF in biological systems should be approached with caution. It has also been demonstrated that the photoreduction of DCF results in the formation of the DCF semiquinone free radical (DCF . ), which, under aerobic conditions, is oxidized by oxygen to its parent dye, DCF, concomitantly forming superoxide radical (28). Moreover, it was reported that DCFH, upon reacting with horseradish peroxidase-compound I or -compound II, was oxidized to DCF . , which was subsequently air-ox...