The tRNA(Thr2) isoacceptor of E. coli has a G-A mismatch at positions 27-43. When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E. coli. Moreover, introduction of the base pair (G-C or U-A) at positions 27-43 of this suppressor tRNA reduced its suppressor activity. These results indicate that the G27-A43 mismatch is necessary for full function of tRNA(Thr2).