The oxidation of 2′,7′-dichlorofluorescin (DCFH) to a fluorescent product is currently used to evaluate oxidant stress in cells. However, there is considerable uncertainty as to the enzymatic and nonenzymatic pathways that may result in DCFH oxidation. Iron/hydrogen peroxide-induced DCFH oxidation was inhibited by catalase or by the hydroxyl radical scavenger dimethylsulfoxide; however, superoxide dismutase (SOD) had no effect on DCFH oxidation. The formation of hydroxyl radical (indicated by the oxidation of salicylic acid to 2,3-dihydroxybenzoic acid) was proportional to DCFH oxidation, suggesting that the hydroxyl radical is responsible for the iron/peroxide-mediated oxidation of DCFH. Utilizing a superoxide generating system consisting of hypoxanthine/xanthine oxidase, oxidation of DCFH was unaffected by SOD, catalase or desferoxamine, and stimulated by removing hypoxanthine from the reaction mixture. In contrast, SOD or elimination of hypoxanthine abolished superoxide formation. In addition, potassium superoxide did not support the oxidation of DCFH. Thus, superoxide is not involved in DCFH oxidation. Boiling xanthine oxidase eliminated its concentration-dependent oxidation of 1 μM DCFH, indicating that xanthine oxidase ian enzymatically utilize DCFH as a high affinity substrate. Kinetic studies of the oxidation of DCFH by xanthine oxidase indicated a K _m(app) of 0.62μM. Hypoxanthine competed with DCFH with a K _i(app) of 1.03 mM. These studies suggest that DCFH oxidation may be a useful indicator of oxidative stress. However, other types of cellular damage may produce DCFH oxidation. For example, conditions or chemicals that damage intracellular membranes may release to the cytoplasm oxidases or peroxidases that might use DCFH as a substrate, similar to xanthine oxidase