Antibiotic susceptibility testing of the rickettsial Q fever agent Coxiella burnetii was performed by using persistently infected L929 fibroblast cells. The efficacies of a variety of antibiotics with different metabolic targets were tested and compared. The most effective antibiotics in bringing about the elimination of the parasite from infected cells included several quinolone compounds and rifampin. Of the quinolone compounds tested, difloxacin (A-56619) was the most effective, followed by ciprofloxacin and oxolinic acid. These three quinolones were apparently rickettsiacidal. After 48 h of exposure to microgram amounts of the compounds (ranging from 2 micrograms of difloxacin per ml to 5 micrograms of the other two antibiotics per ml), the number of intracellular parasites markedly declined; after 10 days of treatment, very few intracellular rickettsiae were detected. Rifampin (1 microgram/ml) was also very effective in eliminating the parasites. Some of the 13 other antibiotics tested that were somewhat effective included chloramphenicol, doxycycline, and trimethoprim. The persistently infected L929 cells were found to provide a convenient system for the relatively rapid determination of the susceptibility of C. burnetii to antibiotics.