We report here the ability of peroxynitrite to cause DNA strand breaks and to oxidize cellular thiol groups in viable rat thymocytes. Peroxynitrite was added to rat thymocytes in a phosphate buffer and DNA damage was measured by the fluorescence analysis of DNA unwinding assay. Peroxynitrite causes DNA strand breaks in a dose-dependent fashion. Four hydroxyl radical scavengers, namely mannitol, dimethyl sulfoxide, sodium benzoate, and Trolox, were tested for their ability to protect DNA from oxidative damage by peroxynitrite. Mannitol failed to protect DNA at concentrations at which it would have conferred nearly complete protection from damage by the hydroxyl radical. Strikingly, dimethyl sulfoxide and benzoate, which are more efficient hydroxyl radical scavengers than mannitol, caused an increase in DNA damage. Trolox was the only scavenger, among the four tested here, that was able to protect DNA from oxidative damage by peroxynitrite. We have previously shown that, among the scavengers tested, Trolox is the most effective scavenger of HOONO*, where HOONO* is a reactive form of HOONO that is a more selective oxidant than is the hydroxyl radical (see W. A. Pryor, X. Jin, and G. L. Squadrito, 1994, Proc. Natl. Acad. Sci. USA 91, 11173-11177). Thus, these results are consistent with our earlier observations that oxidations by peroxynitrite involve a reactive intermediate, HOONO*, rather than hydroxyl radicals. Peroxynitrite also oxidized cellular thiols in a dose-dependent fashion. Greater than 90% of the cells exposed to peroxynitrite were still viable for up to 10 min after DNA damage and thiol oxidation had occurred. In conclusion, DNA damage caused by peroxynitrite can be rationalized as caused by a powerful oxidant, HOONO*, which is formed during the decomposition of peroxynitrite to nitrite.