A case of tick-borne encephalitis (TBE) was found in a farming area located in the southern part of Hokkaido, Japan, in 1993. TBE viruses were isolated from sentinel dogs in the area where the human case occurred in 1995. Ticks were collected in the area by 2 collection methods in 1995 and virus isolation was conducted on ticks in 1995 and 1996. Ixodes ovatus Neumann was found to be the predominant tick species in the area. In 1996,2 virus strains were isolated from 600 I. ovatus ticks (300 females and 300 males), giving the minimum infection rate of 0.33% (2 of 600). The 2 virus strains were identified as TBE virus by antigenic analysis using monoclonal antibodies and the indirect immunofluorescent antibody test. The results showed that I. ovatus was a suspected vector of the emerging TBE virus in Hokkaido.