The wild-type and mutant-type human mitochondrial tRNALeu(UUR) genes were synthesized and transcribed in vitro with T7 RNA polymerase. The kinetic parameters of human mitochondrial leucyl-tRNA synthetase(mtLeuRS) were determined with wild-type and mutant-type human mitochondrial tRNALeu(UUR) respectively. The results show that the value of Km/Kcat of mtLeuRS for the mutant-type tRNALeu(UUR) is 63.9% as compared with the wild-type. Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce it's aminoacylation activity, suggesting it would be one of the mechanisms that the mutation could produce such clinical phenotypes.