The nucleotides in Escherichia coli tRNA(Phe) required for recognition by its cognate synthetase have been determined in vitro by measuring the kinetic parameters for aminoacylation using mutant tRNA(Phe) transcripts with purified E. coli tRNA(Phe) synthetase. The substitution of 11 nucleotides in E. coli tRNA(Phe) is shown to decrease the kcat/KM by as much as 1000-fold relative to the wild type. The most important recognition elements are the three anticodon nucleotides G34, A35, and A36. The recognition set also includes nucleotides in the variable pocket (U20 and U59), the acceptor end (A73), and the tRNA central core (G10, C25, A26, G44, and U45). Many of the recognition nucleotides are also among the residues comprising the identity set determined in vivo using an amber suppressor tRNA(Phe) [McClain, W. H., & Foss, K. (1988) J. Mol. Biol. 202, 697-709]. As could be anticipated from the very different methods used, some nucleotides in the identity set determined by the suppressor method were not among the recognition nucleotides and vice versa. The E. coli tRNA(Phe) recognition data can also be compared to the recognition sets for yeast and human tRNA(Phe) determined previously. The results indicate that the mechanism by which phenylalanyl-tRNA synthetases recognize their substrates seems to have diverged somewhat among different species. For example, nucleotide 20 in the D-loop, the anticodon nucleotides and the discriminator base 73 are important for the recognition by all three enzymes. However, recognition of the tRNA central core nucleotides is unique to E. coli FRS.(ABSTRACT TRUNCATED AT 250 WORDS)