The spectrum of antibiotic susceptibility of Borrelia burgdorferi has been only partially defined. In the present study the effectiveness of 12 antimicrobials, belonging to six different antibiotic classes have been tested against Borrelia burgdorferi s.s. (N=3), Borrelia garinii (N=3), Borrelia afzelii (N=3), Borrelia valaisiana (N=1), and Borrelia bissettii (N=1) isolates. These isolates were analysed by a new standardised colorimetric minimal inhibitory concentration (MIC) method based upon colour changes that result from actively metabolizing spirochaetes after 72 h of incubation. Piperacillin (MIC90: 0.08 mg/l), ceftriaxone (MIC90: 0. 04 mg/l), cefotaxime (MIC90: 0.15 mg/l), azithromycin (MIC90: 0.015 mg/l), roxithromycin (MIC90: 0.05 mg/l) and quinupristin/dalfopristin (MIC90: 0.12 mg/l) gave the lowest MIC values. Minimal inhibibitory activity of amoxycillin (MIC90: 1.04 mg/l), cefixime (MIC90: 1.33 mg/l), cefoperazone (MIC90: 0.83 mg/l) tetracycline (MIC90: 0.29 mg/l) and minocycline (MIC90: 0.30 mg/l) was slightly lower, whereas borrelia were resistant to amikacin (MIC90: >128 mg/l). Mean minimal borreliacidal concentrations (MBCs) were representatively determined for piperacillin (MBC: 1.8 mg/l), ceftriaxone (MBC: 2.0 mg/l), azithromycin (MBC: 0.82 mg/ml), roxithromycin (MBC: 1.8 mg/l), quinupristin/dalfopristin (MBC: 5.0 mg/l), minocycline (MBC: 5.8 mg/l), and amikacin (MBC: >128 mg/l) by using conventional subculture for three weeks in combination with dark-field microscopy. B. garinii proved to be the most susceptible of the genospecies tested. Our study showed excellent in vitro antimicrobial activity of all classes of antibiotics tested, except the aminoglycosides and hence their suitability for therapy of Lyme disease.