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Five mutations in thrS, the gene for threonyl-tRNA synthetase, have been characterized, and the sites of the mutations have been localized to different regions of the thrS gene by recombination with M13 phage carrying portions of the thrS gene. Quantitative immunoblotting shows that some of these mutations cause the overproduction of structurally altered threonyl-tRNA synthetase in vivo. The amounts of in vivo thrS mRNA as measured by quantitative hybridization are, however, the same as wild-type levels for each mutant. These results demonstrate that the expression of threonyl-tRNA synthetase is autoregulated at the posttranscriptional level in vivo.