All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNA(Trp)) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile) and tRNA(Lys) were mainly mitochondrial; and tRNA(Trp) and tRNA(Val) were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNA(Ile) underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNA(Ile). 5'-Extended precursor tRNA(Trp) was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNA(Trp) was imported in vitro more efficiently than edited tRNA(Trp), suggesting the presence of importation determinants in the anticodon.