Defects in mitochondrial function are responsible for many human diseases. Two major problems have hindered gene therapy of mitochondria defects: 1) Mitochondria are in all tissues of the body so a gene therapy must be capable of reaching all tissues. 2) The therapy must localize specifically to mitochondria. Using a short peptide sequence derived from the HIV genome, called TAT, the hypothesis that TAT fusion proteins could cross both mitochondrial membranes and incorporation of a mitochondrial signal sequence (MTS) into a TAT fusion protein would allow processing and localization of exogenous proteins in mitochondria was tested. Using isolated mitochondria, cultured cells, animals, and model membranes, TAT-MTS proteins rapidly transduces into large unilamellar vesicles, cells and mitochondria, and persisted over time in vitro and in vivo. TAT-MTS fusion proteins will be a useful tool in understanding mitochondrial function as well as the development of mitochondrial protein therapies.