Coumermycin A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes supercoiling of DNA and is required for bacterial DNA replication. We have investigated the activity of this coumarin antibiotic on Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease. B. burgdorferi was more susceptible than many other eubacteria to coumermycin as well as novobiocin, another coumarin antibiotic; this contrasted with its relative resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic acid, and ciprofloxacin. Coumermycin at 0.2 micrograms/ml inhibited the growth of B. burgdorferi B31 in BSK II medium. A 100-fold-lower concentration induced the relaxation of two negatively supercoiled circular plasmids within 2 h. Plasmid supercoiling was restored within 2 h of removal of coumermycin. These results suggest that B. burgdorferi has a DNA gyrase and that this enzyme's activity is required for growth. Furthermore, structural analogs of coumermycin may be considered as treatments for Lyme disease.