Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, has been reported to be susceptible to a variety of antimicrobial agents. In this investigation, the action of vancomycin, a glycopeptide antibiotic not previously known to have activity against spirochetes, against borrelias was examined. The in vitro activity of vancomycin against a variety of strains of B. burgdorferi and one strain of Borrelia hermsii was determined by use of a microdilution MIC method (L.L. Dever, J.H. Jorgensen, and A.G. Barbour, J. Clin. Microbiol. 30:2692-2697, 1992). MICs ranged from 0.5 to 2 micrograms/ml. MICs of the glycopeptides ristocetin and teicoplanin and the lipopeptide daptomycin against strain B31 of B. burgdorferi were all > or = 8 micrograms/ml. Subsurface plating, time-kill studies, synergy studies, and electron microscopy were used to investigate further the activity of vancomycin against B31. The MBC of vancomycin was 2 micrograms/ml. Time-kill curves demonstrated > or = 3-log10-unit (99.9%) killing of the final inoculum after 72 h by vancomycin concentrations twice the MIC. Synergy between vancomycin and penicillin was demonstrated at concentrations one-fourth the MIC of each drug. In electron microscopy, B31 cells exposed to vancomycin showed a disruption of cellular integrity and were indistinguishable from those exposed to penicillin. These studies demonstrate another class of microorganisms susceptible in vitro to vancomycin.