Editing reactions are an essential part of biological information transfer processes that require high accuracy, such as replication, transcription, and translation. The editing in amino acid selection for protein synthesis by an aminoacyl-tRNA synthetase, the first proofreading process discovered in the flow of genetic information, prevents attachment of incorrect amino acids to tRNA. Of numerous editing reactions studied in vitro, only one, editing of homocysteine by methionyl-tRNA synthetase, has also been demonstrated in vivo. It is therefore unclear to what extent editing of errors is physiologically relevant. Here we show that isoleucyl-and leucyl-tRNA synthetases also edit homocysteine by cyclizing it to homocysteine thiolactone in the bacterium Escherichia coli. These and other data also suggest that metabolite compartmentation or channeling governs which synthetase participates in editing in bacterial cells.