Variants with mutations in three parts of the tRNA-like structure of turnip yellow mosaic virus RNA (the anticodon, the discriminator position in the amino acid acceptor stem, and in the variable loop) were created via site-directed mutagenesis of a cDNA clone and transcription with T7 RNA polymerase. The valylation properties of transcripts were studied in the presence of pure yeast valyl-tRNA synthetase. Mutation of the central position of the anticodon triplet resulted in a quasi-total loss of valylation activity, indicating that the anticodon is a principal determinant for valylation of the turnip yellow mosaic virus tRNA-like structure. These anticodon mutants interacted with yeast valyl-tRNA synthetase with affinities comparable to those of the wild-type RNA and behaved as competitive inhibitors in the valylation reaction of yeast tRNAVal. The defective aminoacylation of these mutants therefore results from kinetic rather than affinity effects. Minor negative effects on valylation efficiency were observed for mutants with substitutions at the two other sites studied, suggesting a structural role or a limited contribution to the valine identity of the tRNA-like molecule.