A broth microdilution assay was performed to determine the minimal inhibitory concentrations (MICs) of 25 antimicrobial agents for two strains of Borrelia (B.) burgdorferi sensu lato and one strain of B. hermsii. The method comprised BSK II medium lacking gelatin and an incubation period of 72 hours. To investigate the influence of reading mode and density of inoculum on MIC values, microscopical as well as macroscopical MIC reading was performed using standardized final inocula of 10(6) and 10(7) borreliae/ml. Data were processed by two-way analysis of variance. In the microdilution assay, MIC values were significantly influenced either by the inoculum density or reading mode. However, using clearly defined criteria for macroscopical endpoint determination, MICs from macroscopical and microscopical reading were found to be in close agreement. B. burgdorferi sensu lato strains tested were highly susceptible to azithromycin, erythromycin, mezlocillin, piperacillin as well as ceftriaxone, with MICs ranging from < or = 0.016 to 0.125 microgram/ml. B. hermsii was highly susceptible to azithromycin and erythromycin. In comparison to B. hermsii, the beta-lactam antibiotics revealed a significantly higher activity and gentamicin, ofloxacin, and rifampin revealed a significantly lower activity against B. burgdorferi sensu lato strains. To further investigate interactions between BSK II medium, incubation time, and antibiotic efficacy, an agar diffusion bioassay was performed. Out of seven antibiotics tested, the activities of mezlocillin, penicillin G, and piperacillin were significantly influenced by BSK II medium and incubation period and showed a marked decrease of on average 84.0% within 72 hours of incubation.