Recent genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and archaea. The survival of microorganisms missing one or both of these essential enzymes implies an alternative pathway for the formation of Gln-tRNA(Gln) and Asn-tRNA(Asn). This indirect pathway involves misacylation of tRNA(Gln) with Glu (or tRNA(Asn) with Asp) by a nondiscriminating synthetase and subsequent transamidation of the misacylated aa-tRNA by an amidotransferase. Analogues of the natural product puromycin have been developed to inhibit this class of enzymes which has been only recently investigated at the mechanistic and structural levels. The widespread use of the indirect transamidation pathway among prominent human pathogens and its absence in the cytosol of mammalian cells identify aminoacyl-tRNA amidotransferases as an interesting target for the development of antimicrobial agents with a novel mode of action. These mechanism-based inhibitors will provide useful probes for further mechanistic investigations and ligands for X-ray crystallography.