In the yeast Saccharomyces cerevisiae , one of the two cytoplasmic lysine tRNAs, tRNA CUU Lys , is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires binding to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and aminoacylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNA mnm Lys 5 s 2 UUU {where 5-[(methylamino)-methyl]-2-thiouridine is mnm 5 s 2 U} is exclusively localized in the cytoplasm. To study import determinants within the tRNA CUU Lys molecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNA CUU Lys with that of tRNA mnm Lys 5 s 2 UUU abolished import without affecting aminoacylation. A version of tRNA mnm Lys 5 s 2 UUU with an anticodon CUU was efficiently imported in vitro and was also found to be imported in vivo . This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNA CUU Lys transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNA CUU Lys is imported as a folded molecule.