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We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 µg/mL), azithromycin (80 µg/mL), doxycycline (20 µg/mL), or chloramphenicol (100 µg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.