Antibiotic susceptibility testing of two isolates of the Q-fever agent, Coxiella burnetii, was performed with recently and persistently infected L929 fibroblast cells. The two genetically distinct isolates, Nine Mile and Priscilla, are implicated in two different clinical disease syndromes, acute and chronic Q fever, respectively. We compared the efficacies of rifampin, doxycycline, and five 4-quinolone compounds (ciprofloxacin, difloxacin, ofloxacin, norfloxacin, and pefloxacin) in reducing persistent C. burnetii infection of L929 fibroblasts. In persistently infected cells, the Priscilla isolate was less susceptible to all antibiotics tested when compared with the Nine Mile isolate. The most effective antibiotics against the Priscilla isolate were ofloxacin, pefloxacin, and ciprofloxacin (50% inhibitory concentrations of 0.5, 2.2, and 2.5 micrograms/ml, respectively). In persistently infected cells, the Nine Mile isolate was highly susceptible to all antibiotics tested except doxycycline. In contrast, the Priscilla and Nine Mile isolates in recently infected cells were somewhat susceptible to doxycycline; the Priscilla isolate was significantly more susceptible to ofloxacin and rifampin in recently infected host cells than in persistently infected cells. Persistently infected L929 cells were also treated with antibiotic combinations. Although ciprofloxacin and doxycycline had no synergistic effect on the Priscilla isolate, ciprofloxacin and rifampin acted synergistically. Collectively, these in vitro results are in accord with the fact that chronic Q fever in humans is generally not successfully managed with antibiotics. They also indicate that early diagnosis may be essential and that combination antibiotic therapy that includes quinolones may be effective in treating chronic Q fever.