Mitochondrial import of tRNA is now considered as a quasi-universal phenomenon. In the yeast Saccharomyces cerevisiae, one of the three lysine isoacceptors, the tRNA(Lys)1 with the anticodon CUU (tRNA-K1), is encoded by the nuclear genome and distributed between the cytoplasmic (> 95%) and mitochondrial (< 5%) compartments. In vivo and in vitro import assays were developed to study the mechanisms of tRNA-K1 mitochondrial import. Transmembrane translocation of the tRNA requires the intactness of at least two of the components of the mitochondrial import machinery of pre-proteins, MOM19 and MIM44, as well as energy of ATP hydrolysis and an electrochemical potential across the inner membrane. The import of tRNA-K1 involves formation of an RNP complex on the mitochondrial outer membrane. tRNA-K1 import is also dependent upon cytosolic protein factors, one of which was identified as the precursor of the mitochondrial lysyl-tRNA synthetase (MSK). Although essential for tRNA-K1 import in vitro and in vivo, pre-MSK is however not sufficient to direct the import in vitro, which suggests the need of additional cytosolic factor(s). The tRNA can be imported in its mature form and nucleoside modification is not essential. Aminoacylation of the imported tRNA by the cytoplasmic lysyl-tRNA synthetase is a prerequisite for import. Possible mechanisms of intracellular partitioning and mitochondrial membrane translocation of tRNA-K1 are discussed.