||The accuracy of the tRNA aminoacylation, ensured by cognate aminoacyl-tRNA synthetase, is of first importance in all living cells. To understand the mechanism underlying the specific recognition of tRNATrp by tryptophanyl-tRNA synthetase (TrpRS) from three biological domains, Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNATrp, representing the three biological domains, were hyperexpressed in Escherichia coli, and examined regarding their efficiency as substrates for tryptophanylation by recombinant B. subtilis, A . fulgidus, and human TrpRS. Identity elements in tRNATrp contribute to the efficient and specific recognition by cognate TrpRS. To determine the identity elements in tRNATrp, tRNATrp mutants were constructed by site-directed mutagenesis. In B. subtilis tRNATrp, A26 was determined to be a negative identity element and U11-A24, G29-C41, and U31-A39 were determined to be minor identity elements through in vitro tryptophanylation assays. Of these tRNATrp mutants, U11C-A24G and A26G displayed significant structural change in the core region of tRNATrp, revealed by NMR study. The U11C-A24G also affected the expression level of mutant tRNA. In bovine tRNATrp, mutations in either the discriminator base (A73G) or first base pair (G1A-C72U) of acceptor stem of bovine tRNATrp, resulted in a drastic loss of tryptophanylation efficiency, suggesting that A73 and G1-C72 were major positive identity elements in the eukaryotic tRNATrp. The synergetic effects between these two elements suggested a crucial domain required for effective recognition by human TrpRS. The increase of charging ability for G26A suggests that G26 is a negative identity element in bovine tRNATrp. In A . fulgidus tRNATrp, A73 also serves as a major positive identity element, which resembles that of eukaryotic tRNATrp. Like tRNATrp from the other two kingdoms, the G26A mutant of A . fulgidus tRNATrp exhibited increased activity. Therefore, N26 exerts a large influence on tRNATrp tryptophanylation and serves as a negative identity element towards cognate TrpRS. Since the discriminator base (N73) is a major identity element in tRNATrp, the recognition patterns of N73 by TrpRS from the three kingdoms were compared. Results revealed two distinct preference profiles regarding the N73 of tRNATrp substrates: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS based on the kcat/KM values. The preference for G73 in tRNATrp by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNATrp from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria.