Four mutants from Oryza sativa mitochondria tRNA^Trp toward B． subtilis tRNA^Trp were constructed and transcribed in vitro with T7 RNA polymerase. The kinetic parameters (K_cat/K_M) of B． subtilis tryptophanyl-tRNA synthetase(TrpRS) and human TrpRS were determined with four mutant-type tRNA^Trps. Results showed that for reaction with B． subtilis TrpRS, C2/G71 and C4/G69 mutations each induced a comparable 40-fold and 53- fold of activity to Oryza sativa mitochondria tRNA^Trp respectively. Notably, when the C2/G71 and C4/G69 mutations were introduced together into B． subtilis tRNA^Trp, a 140-fold of reaction rate resulted, the catalytic efficiency was 34 percent as that of wild-type B． subtilis tRNA^Trp, but these four mutants resulted in a weak aminoacylation efficiency by human TrpRS, and the change was little. Clearly, the results indicate that C2/G71 and C4/G69 bases in the acceptor stem are important species-specific elements of Oryza sativa mitochondria tRNA^Trp, since which are significant to the aminoacryl activity.