Mitochondria are continually exposed to oxidative stress due to superoxide formation by the respiratory chain which increases in pathological situations such as ischemia reperfusion and neurodegeneration. During oxidative stress there are a number of changes in mitochondrial low-molecular-weight and protein thiols. In particular, the mitochondrial glutathione pool becomes oxidized and forms mixed disulfides with protein thiols. To investigate changes in the redox state and conjugation of mitochondrial glutathione, and other mitochondrial thiols, we designed and characterized a thiol probe specifically targeted to the mitochondrial matrix. This molecule, thiobutyltriphenylphosphonium bromide, contains a thiol group linked to a lipophilic triphenylphosphonium cation which causes it to accumulate in the negatively charged mitochondrial matrix. Using [14C]thiobutyltriphenylphosphonium bromide we confirmed that it was selectively accumulated by isolated mitochondria. In the mitochondrial matrix the thiol group equilibrated with endogenous thiols and during oxidative stress became disulfide-bonded to protein and nonprotein thiols. Therefore, this novel thiol probe can be used to label protein thiol groups and to investigate changes in conjugation and redox state of mitochondrial thiols during oxidative stress.