A growing number of mutated mitochondrial tRNA genes have been found associated with severe human diseases. To investigate the potential interference of such mutations with the primordial function of tRNAs, i.e. their aminoacylation by cognate aminoacyl-tRNA synthetases, a human mitochondrial in vitro aminoacylation system specific for isoleucine has been established. Both native tRNAIleand isoleucyl-tRNA synthetase activity have been recovered from human placental mitochondria and the kinetic parameters of tRNA aminoacylation determined. The effect of pathological point mutations present in the mitochondrial gene encoding tRNAIlehas been tackled by investigating the isoleucylation properties of wild-type and mutated in vitro transcripts. Data show that: (i) modified nucleotides contribute to efficient isoleucylation; (ii) point mutation A4269G in the gene (A-->G at nt 7 in the tRNA), associated with a cardiomyopathy, does not affect aminoacylation significantly; (iii) point mutation A4317G (A-->G at nt 59 in the tRNA), reported in a case of fatal infantile cardiomyopathy, induces a small but significant decrease in isoleucylation. The potential implications of these findings on the understanding of the molecular mechanisms involved in the expression of pathology are discussed.