In May 1993, we collected unfed ticks in Anan, Tokushima Prefecture, Shikoku District, Japan, a highly endemic area of Japanese spotted fever (JSF). We isolated JSF rickettsiae from these ticks by inoculating tick-emulsions into L-929 cells. Of the four genera and seven species of ticks examined, only one larva of Dermacentor taiwanensis Sugimoto yielded a rickettsial isolate. The isolate (DT-1 strain) was found to belong to the serotype common to the causative agent of JSF. This was based on its specific reactivity to mouse antisera and to a monoclonal antibody to JSF rickettsiae from the Shikoku District. This is the first tick-borne spotted fever group of rickettsia isolated in Japan. However, all stages of D. taiwanensis infrequently parasitize humans. Some ticks in the genus Haemaphysalis commonly infest humans and are frequently found to be rickettsia-positive in the hemolymph test when using the monoclonal antibody mentioned above. Therefore, the vectors of JSF rickettsiae to humans may be a complex of tick species.