Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.