An analogue of isoleucyl-adenylate (Ile-AMS) potently inhibits the isoleucyl-tRNA synthetases (IleRSs) from the three primary kingdoms, whereas the antibiotic mupirocin inhibits only the eubacterial and archaeal IleRSs, but not the eukaryotic enzymes, and therefore is clinically used against methicillin-resistant Staphylococcus aureus. We determined the crystal structures of the IleRS from the thermophilic eubacterium, Thermus thermophilus, in complexes with Ile-AMS and mupirocin at 3.0- and 2.5-A resolutions, respectively. A structural comparison of the IleRS.Ile-AMS complex with the adenylate complexes of other aminoacyl-tRNA synthetases revealed the common recognition mode of aminoacyl-adenylate by the class I aminoacyl-tRNA synthetases. The Ile-AMS and mupirocin, which have significantly different chemical structures, are recognized by many of the same amino acid residues of the IleRS, suggesting that the antibiotic inhibits the enzymatic activity by blocking the binding site of the high energy intermediate, Ile-AMP. In contrast, the two amino acid residues that concomitantly recognize Ile-AMS and mupirocin are different between the eubacterial/archaeal IleRSs and the eukaryotic IleRSs. Mutagenic analyses revealed that the replacement of the two residues significantly changed the sensitivity to mupirocin.