Abstract: Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.