We have isolated mutations in the Escherichia coli glnS gene encoding glutaminyl-tRNA synthetase [GlnS; L-glutamine:tRNAGln ligase (AMP-forming), EC 6.1.1.18] that give rise to gene products with altered specificity for tRNA and are designated "mischarging" enzymes. These were produced by nitrosoguanine mutagenesis of the glnS gene carried on a transducing phage (lambda pglnS+). We then selected for mischarging of su+3 tRNATyr with glutamine by requiring suppression of a glutamine-requiring beta-galactosidase amber mutation (lacZ1000). Three independently isolated mutants (glnS7, glnS8, and glnS9) were characterized by genetic and biochemical means. The enzymes encoded by glnS7, glnS8, and glnS9 appear to be highly selective for su+3 tRNATyr, because in vivo mischarging of other amber suppressor tRNAs was not detected. The GlnS mutants described here retain their capacity to correctly aminoacylate tRNAGln. All three independently isolated mutant genes encode proteins with isoelectric points that differ from those of the wild-type enzyme but are identical to each other. This suggests that only a single site in the enzyme structure is altered to give the observed mischarging properties. In vitro aminoacylation reactions with purified GlnS7 protein show that this enzyme can also mischarge some tRNA species lacking the amber anticodon. This is an example of mischarging phenotype conferred by a mutation in an aminoacyl-tRNA synthetase gene; the results are discussed in the context of earlier genetic studies with mutant tRNAs.