The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition. All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus. Thus, the recognition by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA. The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro. Here, we have determined the aminoacylation level in vivo of tRNA^Glu, tRNA^Lys, and tRNA₁^GIn in Escherichia coli strains containing undermodified derivatives of mnm⁵s²U34. Lack of the 5-methylaminomethyl group did not reduce charging levels for any of the three tRNAs. Lack of the s²U34 modification caused a 40% reduction in the charging level of tRNA^Glu. Charging of tRNA^Lysand tRNA₁^Gln were less affected. There was no compensating regulation of expression of glutamyl-tRNA synthetase because the relative synthesis rate was the same in the wild-type and mutant strains. These results indicate that the mnm⁵U34 modification is not an important recognition element in vivo for the glutamyl-tRNA synthetase. In contrast, lack of the s²U34 modification reduced the efficiency of charging by at least 40%. This is the minimal estimate because the turn-over rate of Glu-tRNA^Glu was also reduced in the absence of the 2-thio group. Lack of either modification did not affect mischarging or mistranslation.