Nitric oxide reacts with superoxide to form peroxynitrite, which may be an important mediator of free radical-induced cellular injury. Oxidation of dihydrorhodamine to fluorescent rhodamine is a marker of cellular oxidant production. We investigated the mechanisms of peroxynitrite-mediated formation of rhodamine from dihydrorhodamine. Peroxynitrite at low levels (0-1000 nM) induced a linear, concentration-dependent, oxidation of dihydrorhodamine. Hydroxyl radical scavengers mannitol and dimethylsulfoxide had minimal effect (< 10%) on rhodamine production. Peroxynitrite-mediated formation of rhodamine was not dependent on metal ion catalyzed reactions because studies were performed in metal ion-free buffer and rhodamine formation was not enhanced in the presence of Fe3+ ethylenediaminetetraacetic acid (EDTA). Thus, rhodamine formation appears to be mediated directly by peroxynitrite. Superoxide dismutase slightly enhanced rhodamine production. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dihydrorhodamine oxidation through competetive oxidation of free sulfhydryls. Urate was also an efficient inhibitor (KI approximately 2.5 microM), possibly by reduction of an intermediate dihydrorhodamine radical and recycling of dihydrorhodamine. Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine. In the presence of oxygen, nitric oxide induces a relatively slow oxidation of dihydrorhodamine due to the formation of nitrogen dioxide. We conclude that dihydrorhodamine is a sensitive and efficient trap for peroxynitrite and may serve as a probe for peroxynitrite production.