Rhodamine 123, a mitochondrial stain that preferentially accumulates in certain cancer cells, has been reduced and iodinated by using NaI in the presence of N-chlorosuccinimide. The various mono-, di-, and triiodo derivatives have been isolated and characterized. These nonfluorescent compounds are taken up by mammalian cells, become fluorescent within the cytoplasm (presumably following oxidation), and show the same pattern of localization as the parent compound. Iodination with no-carrier-added Na125I yields the same mixture of compounds. All 125I derivatives accumulate preferentially in PC3 adenocarcinoma cells compared with V79 lung fibroblasts, with the differential being greatest for the monoiodo compound, followed by the di- and triiodo derivatives.