A single form of tRNA(Trp) exists in the yeast cytoplasm to respond to the unique codon, UGG, which specifies this amino acid. Mutations in the anticodon of the corresponding gene, which generate potential nonsense suppressor tRNAs, have been generated in vitro and tested in vivo for biological activity. The amber (C35U) and opal (C34U) suppressors show strong and weak activities respectively while the ochre suppressor (C34U,C35U) has no detectable biological activity. To understand the basis for these differences, a set of synthetic tRNA(Trp) genes has been constructed to permit in vitro, T7 RNA polymerase synthesis of transcripts corresponding to the normal and mutant tRNAs. Kinetic parameters for aminoacylation of these transcripts by purified, yeast, tryptophanyl-tRNA synthetase have been measured and compared to values observed using the naturally occurring tRNA(Trp) as a substrate. The efficiency of aminoacylation is reduced by 40, 2000, and 30,000 fold by the C35U, C34U, and C34U,C35U mutations respectively. Interestingly, the C35U change affects only tRNA binding while C34U also alters catalytic efficiency. We conclude that both C34 and C35 are major identity elements in the recognition of tRNA(Trp) by its cognate synthetase. These differences in aminoacylation efficiency closely parallel the in vivo suppressor activities of the mutants.