Fourteen mutant strains of Escherichia coli were examined, each of which requires tryptophan for growth but is unaltered in any of the genes of the tryptophan biosynthetic operon. The genetic lesions responsible for tryptophan auxotrophy in these strains map between str and malA. Extracts of these strains have little or no ability to charge transfer ribonucleic acid (tRNA) with tryptophan. We found that several of the mutants produce tryptophanyl-tRNA synthetases which are more heat-labile than the enzyme of the parental wild-type strain. Of these heat-labile synthetases, at least one is protected against thermal inactivation by tryptophan, magnesium, and adenosine triphosphate. Two other labile synthetases which are not noticeably protected against heat inactivation by substrate have decreased affinity for tryptophan. On low levels of supplied tryptophan, these mutants exhibit markedly decreased growth rates but do not contain derepressed levels of the tryptophan biosynthetic enzymes. This suggests that the charging of tryptophan-specific tRNA is not involved in repression, a conclusion which is further substantiated by our finding that 5-methyltryptophan, a compound which represses the tryptophan operon, is not attached to tRNA by the tryptophanyl-tRNA synthetase of E. coli.