The specificity of the tyrosyl-tRNA synthetases from Escherichia coli and bacillus stearothermophilus for tyrosine compared with phenylalanine has been determined by using samples of phenylalanine which have been scrupulously freed from tyrosine by either chemical or enzymic scavenging procedures. Both kinetic measurements and product analyses give a value of 1 x 10(5)-2 x 10(5) for the preferential activation of tyrosine. Combined with the known ratio of phenylalanine to tyrosine in rapidly growing E. coli, an error rate of about approximately 5/10(4) is calculated for the misactivation of phenylalanine. Since we find no evidence for an editing mechanism and this error rate is similar to observed rates in protein synthesis, the tyrosyl-tRNA synthetases appear to have adequate amino acid selection by simple preferential binding of the correct substrate. The incremental binding energy of the phenolic hydroxyl group of tyrosine is approximately 7 kcal/mol, a value presumed close to the maximum possible because of the evolutionary pressure on tyrosyl-tRNA synthetases for maximum specificity. A summary of high incremental binding energies determined from experiments on aminoacyl-tRNA synthetase is presented.