To determine the vertebrate host of tick-borne encephalitis (TBE) virus in the southern part of Hokkaido, Japan, virus isolation was performed using spleens from small mammals captured in the area. Two virus strains were isolated, one strain from Apodemus speciosus and another from Clethrionomys rufocanus. Virus isolates were inoculated onto baby hamster kidney cell monolayers and antigen slides were prepared for an indirect immunofluorescent antibody assay. Two isolates were identified as TBE viruses by monoclonal antibody reactions. To specify the TBE-endemic area in Hokkaido, rodent, horse, and dog sera collected from 1992 to 1997 were tested for neutralization antibody against TBE virus previously isolated from a dog. The positive cases were distributed in four districts in the southern part of Hokkaido.