In Saccharomyces cerevisiae, one of the two cytoplasmic lysine-tRNA isoacceptors, tRNA(LysCUU), is the only nuclear-coded tRNA species to be mitochondrially-imported. Using an in vitro assay of tRNA import, we have studied if the mitochondrial protein translocation machinery is also involved in the mitochondrial targeting of this tRNA. As for protein import, tRNA import also requires the integrity of proteins exposed onto the mitochondrial outer membrane and is dependent upon energy and membrane eletrochemical potential. Furthermore, partial ATP-depletion of the import system leads to stabilization of a translocation intermediate containing the tRNA. In this case, the import of the tRNA can be completed after addition of a new source of energy, even without restoration of the membrane electrochemical potential and without further addition of cytoplasmic proteins. Outer and inner mitochondrial membrane receptors shown to participate in translocation of mitochondrial pre-proteins (MOM19 and MPI1/MIM44, respectively) are also important for import of the tRNA, in vitro as well as in vivo. In contrast, the outer membrane receptor MOM72 is dispensable for the import. Taken together, these results demonstrate that a functional mitochondrial protein translocation apparatus is required for the mitochondrial import of the yeast tRNA(LysCUU).