Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.