We report here the cloning and primary structure of Mycobacterium tuberculosis isoleucyl-tRNA synthetase. The predicted 1035-amino acid protein is significantly more similar in sequence to eukaryote cytoplasmic than to other eubacterial isoleucyl-tRNA synthetases. This similarity correlates with the enzyme being resistant to pseudomonic acid A, a potent inhibitor of Escherichia coli and other eubacterial isoleucyl-tRNA synthetases, but not of eukaryote cytoplasmic enzymes. Consistent with its eukaryote-like features, and unlike E. coli isoleucyl-tRNA synthetase, the M. tuberculosis enzyme charged yeast isoleucine tRNA. In spite of these eukaryote-like features, M. tuberculosis isoleucyl-tRNA synthetase exhibited highly specific cross-species aminoacylation, as demonstrated by its ability to complement isoleucyl-tRNA synthetase-deficient mutants of E. coli. When introduced into a pseudomonic acid-sensitive wild-type strain of E. coli, the M. tuberculosis enzyme conferred trans-dominant resistance to the drug. The results demonstrate that the sequence of a tRNA synthetase could have predictive value with respect to the interaction of that synthetase with a specific inhibitor. The results also demonstrate that mobilization of a pathogen's gene for a drug-resistant protein target can spread resistance to other, normally drug-sensitive pathogens infecting the same host.