Abstract: Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase were examined using in vitro transcripts. By mutation studies, a marked decrease in aminoacylation with threonine showed that the first base pair in the acceptor stem and the second and third positions of the anticodon are major identity elements of tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). Base substitution of the discriminator base, A73, by G73 or C73 impaired the threonine accepting activity, but not that by U73, suggesting that this position contributes to discrimination from other tRNAs possessing G73 or C73. No effects on aminoacylation were observed with substitutions at the second base pair in the acceptor stem. These are in contrast to E.coli tRNA(Thr) where the second base pair is required for the specific aminoacylation, with the discriminator base playing no roles. Of several mutations at the third base pair in the acceptor stem, only the G3-U70 mutation impaired the activity, suggesting that the G3-U70 wobble pair, the identity determinant of tRNAAla, acts as a negative element for threonyl-tRNA synthetase. These findings indicate that while the first base pair in the acceptor stem and the anticodon nucleotides have been retained as major recognition sites between S. cerevisiae and E.coli tRNA(Thr), the mechanism by which the synthetase recognizes the vicinity of the top of the acceptor stem seems to have diverged with the species.