The wood mouse (Apodemus speciosus) serves as a wildlife reservoir for Lyme disease spirochetes in Hokkaido, Japan. To isolate Borrelia species, we captured 34 wood mice in an area where Borrelia species are endemic during October 1993. The earlobes (right and left), heart, spleen, and urinary bladder from each mouse were used as culture sources. As a result of culture 73 isolates from 21 mice were classified by rRNA gene restriction fragment length polymorphism analysis. Ribotype groups III (Borrelia afzelii) and IV (unknown species) were detected among those isolates. Thirty-one (77.5%) of 40 earlobe isolates were classified as group IV. In contrast, 6 (40.0%) of 15 heart isolates, 5 (50.0%) of 10 spleen isolates, and 7 (87.5%) of 8 urinary bladder isolates were B. afzelii. Seven mice showed mixed infection with B. afzelii and group IV. The data indicate that different Borrelia species can coexist in a reservoir host that is suitable for them.