In this study, the varying reactivities of Bacillus subtilis tryptophanyl-tRNA synthetase toward prokaryotic, eukaryotic, and halophile tRNAs were employed to define the potential identity elements on tRNA(Trp). On this basis mutagenesis was performed to obtain, through in vivo heterologous expression in Escherichia coli and in vitro transcription with T7 RNA polymerase, mutant B. subtilis tRNA(Trp) for comparison with the wild-type. These comparisons served to establish G73 and the anticodon as major identity elements, and A1-U72, G5-C68, and A9 as minor identity elements. While the tryptophanyl-tRNA synthetase from B. subtilis and E. coli require G73 to function, replacement of G73 by A73 favors the enzyme from yeast. This change points to the variation of the identity elements for the same amino acid among different organisms. The similarity in these elements between B. subtilis and E. coli tryptophanyl-tRNA synthetase, however, suggests that identity elements on tRNA, like the active centers on enzymes, undergo evolutionary change at slower rates than less essential portions of the macromolecule.