Intracellular metabolism of the carcinogen chromate [Cr(VI)] produces the oxidative stress and oxidative DNA damage associated with its genotoxicity. Such oxidative stress has previously been measured by fluorescence using oxidant-sensitive dyes and attributed to the formation of reactive oxygen species (ROS). However, metabolism of Cr(VI) also produces Cr-(IV) and Cr(V) which can directly damage biological macromolecules without forming ROS. We used the high-valence chromium species, bis(2-ethyl-2- hydroxybutyrato)oxochromate(V) [Cr(V)-EHBA], to test whether high-valence chromium would also react with the oxidantsensitive dyes 2',7'- dichlorofluorescin (DCFH) and dihydrorhodamine (DHR). Cr(V)-EHBA caused both dyes to fluoresce over a wide dynamic range and under conditions which indicated that Cr(V) had reacted directly with both dyes without first forming a diffusible radical species. Dimethylthiourea (DMTU) and ethanol did not affect Cr(V)-induced fluorescence in vitro or Cr(VI)-induced fluorescence in A549 cells. Under the same conditions, ethanol and DMTU increased the extent of hydrogen peroxide-induced fluorescence. As chromium-induced fluorescence was unaffected by radical scavengers and was qualitatively different from hydrogen peroxide-induced fluorescence, we conclude that DCF and R123 fluorescence in chromatetreated A549 cells is a qualitative and cumulative measure of intracellular Cr(V) formation and not ROS.