To study the mechanisms of targeting tRNA(CUULys) from the cytoplasm into the mitochondrial compartment of the yeast cells, two test systems have been developed. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm; however, only tRNA(CUULys) was found in the mitochondrial compartment. The in vitro import of this tRNA into isolated mitochondria required the presence of ATP and soluble cellular proteins in the reaction mixture. Two protein fractions were found to be necessary to direct the import in vitro. The first one had a high heparin-binding affinity, while the other one was not retained on heparin-Sepharose. Aminoacylation of the tRNA(Lys) before the transport and/or addition of the correspondent lysyl-tRNA synthetase to the in vitro system increased the efficiency of the import, but the protein fraction with heparin-binding properties is still required. The unmodified transcripts of the tRNA(CUULys) gene were shown to be able to be transported into isolated yeast mitochondria.