A kinetic analysis of aminoacylation of mutant yeast tRNA(Phe) transcripts by its cognate yeast phenylalanyl-tRNA synthetase (FRS) reveals five nucleotides in tRNA(Phe) as major recognition sites for FRS. The aminoacylation kinetics for two double mutants suggest that each of the five recognition sites contributes independently to kcat/KM. Measured kinetic values for the mutants presented here and those reported previously were then used to calculate the predicted kcat/KM of misacylation for a number of noncognate tRNAs. The predicted kcat/KM values are consistent with values measured by other investigators and thus support the five-nucleotide recognition model. The kcat/KM of misacylation for all known yeast tRNAs has been calculated on the basis of this model, and the specificity of FRS for tRNA(Phe) in yeast is discussed.