To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.