A new antibiotic, lysobactin (l), was isolated from fermentations of Lysobacter sp. ATCC 53042. Lysobactin was shown to be a potent agent against Gram-positive aerobic and anaerobic bacteria in vitro, and its efficacy in vivo was found to compare favorably with the clinically useful antibiotic vancomycin. Physicochemical characterization identified the antibiotic as a dibasic peptide of nominal mass 1275 Da. The structure of lysobactin, including stereochemical details, was determined by a combination of chemical and enzymatic degradations that were analyzed primarily by mass spectrometry. On the basis of synthetic modifications, the macrocyclic lactone and N-terminal D-amino acid of lysobactin are important structural elements contributing to the antibacterial activity.