In the yeast Saccharomyces cerevisiae, one of the two cytoplasmic lysine tRNAs, tRNALys/CUU, 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 amino-acylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNALys/mnm5s2UUU (where 5-(methylamino)-methyl-2-thiouridine is mnm5s2U) is exclusively localized in the cytoplasm. To study import determinants within the tRNALys/CUU 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 preMSK. 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 tRNALys/CUU with that of tRNALys/mnm5s2UUU abolished import without affecting aminocylation. A version of tRNALys/mnm5s2UUU 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 tRNALys/CUU transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNALys/CUU is imported as a folded molecule