Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis. We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon. One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U). These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated. The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon.