In Escherichia coli, uridine in the wobble position of tRNA(Glu) and tRNA(Lys) is modified to mnm⁵s²U34. This modification is believed to restrict the base-pairing capability, i.e. to prevent misreading of near-cognate codons and reduce the efficiency of cognate codon reading, especially of codons ending in G. We have determined the influence of the 5-methylaminomethyl and the 2-thio modifications of mnm⁵s²U34 in tRNA(Glu) the on translation rate of the glutamate codons GAA and GAG in vivo. in wild-type cells, GAG is translated slower (7.7 codons/second) and GAA faster (18 codons/second) than the average codon (13 codons/second). Surprisingly, tRNA(Glu) lacking the 5-methylaminomethyl group, thus containing s²U34, translated GAA twofold faster (47 codons/second) and GAG fourfold slower (1.9 codons/second) than fully modified tRNA(Glu). In contrast, tRNA(Glu) that contains mnm⁵U34 instead of mnm⁵s²U34 translated GAA fourfold slower (4.5 codons/second) and GAG only 20% slower (6.2 codons/second). Clearly, the 5-methylaminomethyl group of mnm⁵s²U34 facilitates base-pairing with G while decreasing base-pairing with A, resulting in rates of translation of GAG and GAA that approach that of the average codon. The 2-thio group increases the recognition of GAA and has only a minor effect on the decoding of GAG. Furthermore, the 2-thio group is important for aminoacylation (see the accompanying paper). These data imply that the function of mnm⁵s²U34 may be different from what has been suggested previously.