The evolutionary loss in eukaryotic cells of mitochondrial (mt) tRNA genes and of tRNA structural information in the surviving genes has led to the appearance of mt-tRNAs with highly unusual structural features. One such mt-tRNA is the human mt-tRNALys, which relies on post-transcriptional base modification to achieve correct three-dimensional structure. It has been shown that the in vitro transcript of human mt-tRNALys adopts a particular, non-cloverleaf structure when devoid of modified bases, while the native, fully modified tRNA shows the expected cloverleaf structure. Furthermore, a methyl group at position A9-N1, introduced chemically in an otherwise unmodified mt-tRNALys transcript, was found to induce a stable cloverleaf conformation, raising the question of how the specific methyltransferase recognizes the unmodified transcript. In order to shed light on this unusual case of tRNA maturation, the tRNA modification enzymes contained in protein extracts from either highly purified HeLa cell mitochondria or HeLa cell cytosol were first identified and compared, and then used to analyze the mt-tRNALys. An initial screening for modification activities, using as substrates unmodified in vitro transcripts of tRNA genes with well characterized structures, namely yeast cytosolic tRNAPhe, human cytosolic tRNA3Lys, and human mt-tRNAIle, revealed the presence of nine and 11 modification activities in the mitochondrial and cytosolic protein extracts, respectively, the mitochondrial extract including a tRNA (adenine-9,N1)-methyltransferase activity. The comparison of the level and kinetics of A9-N1 methylation and other secondary modifications in the unmodified, misfolded mt-tRNALys and in a cloverleaf-shaped structural mutant, engineered to adopt the tRNALys cloverleaf structure without post-transcriptional modifications, suggested strongly that the methylation of A9-N1 in tRNALys proceeds via a cloverleaf-shaped intermediate. Therefore, it is proposed that this intermediate is present in the in vitro transcript as part of a dynamic equilibrium, and that the mitochondrial protein extract contains an activity that stabilizes, by secondary modification, such a transient cloverleaf-shaped intermediate. Thus, countering the evolutionary loss of structural information in mt-tRNA genes, the mt-tRNA structure is maintained by a modification enzyme encoded in nuclear DNA.