The aminoacylation kinetics of 19 different variants of yeast tRNATyr with nucleotide substitutions in positions 33-35 were determined. Substitution of the conserved uridine-33 does not alter the rate of aminoacylation. However, substitution of the anticodon position 34 or position 35 reduces Km from 2- to 10-fold and Vmax as much as 2-fold, depending on the nucleotide inserted. The ochre and amber suppressor tRNAsTyr both showed about a 7-fold reduction in Vmax/Km. Data from tRNATyr with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of psi-35. The effect of simultaneous substitutions at positions 34 and 35 can be predicted reasonably well by combining the effects of single substitutions. These data suggest that yeast tyrosyl-tRNA synthetase interacts with positions 34 and 35 of the anticodon of tRNATyr and opens the possibility that nonsense suppressor efficiency may be mediated by the level of aminoacylation.