The set of nucleotides in Escherichia coli tRNA(Gln) which facilitate aminoacylation by glutaminyl-tRNA synthetase (GlnRS) has been defined [Hayase et al. (1992), EMBO J. 11, 4159-4165]. To determine whether the glutamine "identity set" is sufficient to confer acceptance on a noncognate tRNA, we constructed tRNA(Glu) mutants with the set of glutamine recognition elements. These mutants were examined for aminoacylation in vitro with GlnRS and also with glutamyl-tRNA synthetase (GluRS) to correlate gains in glutamine acceptance with losses of glutamate acceptance. Incorporating glutamine recognition elements in only the acceptor stem or anticodon loop of tRNA(Glu) improved the specificity constant (kcat/KM) for aminoacylation by GlnRS. However, the introduction of all defined glutamine recognition elements in tRNA(Glu) resulted in a substrate with a specificity constant 100-fold below that for aminoacylation of tRNA(Gln). Including the tertiary framework of tRNA(Gln) (in addition to the glutamine recognition elements) in the tRNA(Glu) context further improved aminoacylation by GlnRS, but the specificity was still reduced compared with that of tRNA(Gln). The increase in glutamine acceptance was correlated for all mutants with a decrease in glutamate acceptance, indicating that GluRS also recognizes acceptor stem and anticodon sequences in cognate tRNA. The inability to completely convert tRNA(Glu) to glutamine acceptance with these mutations suggests that tRNA(Glu) contains antideterminants to glutamine identity. The analysis of these mutants with both enzymes revealed that there is a strong element of discrimination between glutamate and glutamine tRNAs associated with the anticodon. To test this dependence, mutants of both tRNAs were made to effect anticodon switches to the possible glutamate and glutamine isoacceptors. The kinetic evaluation of the anticodon switch mutants suggests that overlap in anticodon recognition is avoided through specificity for the third anticodon position coupled with divergent preferences for the wobble base.