Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code. They are synthesized by the aminoacyl-tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations. Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Sequence-specific interactions between GlnRS and tRNA(Gln) determine both the accuracy of tRNA selection and the efficiency of aminoacylation. Thus, amino acid recognition is tRNA-dependent. Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA-enzyme complex displays a considerably reduced affinity for glutamine compared to wild-type. This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.