Abstract Mischarging mutants of Escherichia coli sup3 tyrosine transfer RNA have been isolated by selecting for suppression of bacterial amber mutations not suppressed by sup3. Five of the mutants have single base changes in the amino acid acceptor stem (A1, A2, U80, U81 and G82). Mutants A1 and A2 are weak thermosensitive suppressors from which thermostable derivatives have been isolated. Some of these derivatives affect the amount of tRNA synthesized but not the sequence (precursor or promoter mutations), and others are double mutants A1U81 and A2U80. The latter mutant does not mischarge. The efficiency of suppression of A1 and A2 can also be increased by recombination events that lead to duplication and triplication of the suppressor gene. The amino acid inserted by some of these mutants at the amber site has been determined. Mutant A1 inserts glutamine, while U81 and A1U81 insert both glutamine and tyrosine. Taken together the results show that the terminal part of the amino acid acceptor stem has an important role in the specificity of aminoacylation by the glutamine and tyrosine synthetase.