Alterations to the redox state of mitochondrial thiols is a particularly important response to oxidative stress, because mitochondria are a major source of reactive oxygen species within cells. To investigate these changes we designed and synthesized a novel probe for mitochondrial thiols, thiobutyltriphenylphosphonium bromide [TBTP; R. J. Burns, R. A. J. Smith, and M. P. Murphy (1995) Arch. Biochem. Biophys. 322, 60-68]. This lipophilic cation was accumulated several hundred-fold into the negatively charged matrix of isolated mitochondria, where it equilibrated with endogenous thiols and during oxidative stress formed disulfide bonds to exposed protein thiols. In this paper we show that TBTP can also selectively react with mitochondrial protein thiols in living cells. Since TBTP localizes specifically to mitochondria and forms disulfide bonds selectively with mitochondrial proteins during oxidative stress, we conclude that TBTP has utility for investigating changes in mitochondrial thiols, independently of alterations occurring in the cytoplasm.