Mutations of mtDNA, particularly those in mtDNA-encoded tRNA genes, are emerging as a significant cause of human disease. We examined the effects of the pathogenic A3243G and T3271C mutations in the mitochondrial tRNA(Leu(UUR)) gene on the aminoacylation of tRNA(Leu(UUR)). Transmitochondrial cells carrying these mutations have decreased steady-state levels of mitochondrial tRNA(Leu(UUR)). The A3243G mutation also results in a decrease in the fraction of aminoacylated tRNA(Leu(UUR)). To determine if the decreased fraction of aminoacylated tRNA(Leu(UUR)) in A3243G mutant cells was due to a defect in the ability of mutant tRNA to be aminoacylated by the human mitochondrial leucyl-tRNA synthetase, we examined the aminoacylation kinetics of wild-type and mutant tRNA(Leu(UUR)), using both native and in vitro transcribed tRNA(Leu(UUR)). Native A3243G mutant tRNA(Leu(UUR)) was 25-fold less efficiently aminoacylated in vitro, compared to native wild-type tRNA(Leu(UUR)). The T3271C mutation in tRNA(Leu(UUR)) did not affect the efficiency of aminoacylation of the native tRNA. There were no differences in aminoacylation efficiencies among wild-type and mutant tRNA(Leu(UUR)) transcripts. The combined effects of the reductions in the steady-state levels and the aminoacylated fraction of tRNA(Leu(UUR)) are likely to contribute to the decreases in the rates of mitochondrial translation observed in mutant cells. These results also suggest that the A3243G and T3271C mutations may have distinct mechanisms of pathogenesis.