Reduced “leuco” dyes such as dichlorodihydrofluorescein (DCFH2) are widely used as profluorescent probes for oxidative stress, although they require a catalyst to be oxidized by hydrogen peroxide and react indiscriminately with oxidizing radicals and the fluorescent product (DCF) is a potential photosensitizer of superoxide generation. In this study, key properties of the radical intermediate in oxidation (“semiquinone,” DCFH•/DCF•−) were measured, to help understand the reactions that can occur in biological systems. The intermediate was generated by oxidizing DCFH2 or reducing DCF by radiolytically generated radicals and monitoring the reactions using kinetic spectrophotometry. The semiquinone showed pH-sensitive absorption spectral changes, decay kinetics (both in the absence and in the presence of oxygen), and reduction potential, all corresponding to prototropic dissociations with pKa's of ˜7.1 and 9.0. DCFH2 has pKa's in a similar region (8–9) and hence pH variations are potentially important in the use of this probe. The rate constant for reaction of the semiquinone with oxygen at pH 7.4 is 5.3 × 108 M−1 s−1: this reaction, rather than disproportionation of DCFH•/DCF•−, generates DCF in biological systems, concomitantly forming superoxide and hence H2O2 to cycle the catalyst. The midpoint reduction potential of the couple DCF,H+/DCFH• is approximately −0.75 V vs. NHE at pH 7.4; DCF is unlikely to be reduced rapidly by common flavoprotein reductases.