The methylation of 2′-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2′-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon–anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2′-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1Ser, using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2′-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2′-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2′-O-methyl nucleosides are not found in these positions within natural tRNA species.