E. coli methionyl-tRNA synthetase is one of the class I tRNA synthetases. The Tryptophane residue at the position 461 located in the C-terminal dornain of the enzyme is a key amino acid for the interaction with the anticodon of tRNA^(Met). W461 was replaced with other amino acids to determine the chemical requirement for the interaction with the anticodon of tRNA^(Met). Saturation mutagenesis at the position 461 generated a total of 12 substiturion mutants of methionyl-tRNA synthetase. All the mutants showed the same in vivo stability as the wild-type enzyme, suggesting that the amino acid substitutions did not cause severe conformational change of the protein. The mutants containing tyrosine, phenylalanine, histidine and cysteine substitutions showed in vivo activity while all the other mutants did not. The comparison of the in vitro aminoacylation activities of these mutants showed that aromatic ring structure, Van der Waals volume and hydrogen bond potential of the amino acid residue at the position 461 are the major detemunants for the interaction with the anticodon of tRNA^(Met).