Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3' end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3' end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3' end metabolism (3' end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases.