To obtain epidemiological data on human Lyme disease (LD) in Japan, anti Borrelia burgdorferi (Borrelia) antibody was measured in dogs by enzyme-linked immunosorbent assay (ELISA). The anti-Borrelia antibody titer was high in 106 (27.3%) of 387 clinically healthy dogs that may have a chance of tick bite but not in any of 31 negative control dogs not exposed to ticks; the difference of the antibody titer between two groups was significant. However, no signs of arthritis were observed in any of the 106 dogs with a high anti-Borrelia antibody titer. The antibody titer was also significantly differed between the age groups less than 1 year old and 1 year old or more, the group maintained indoors and that maintained outdoors, the group with tick bites and that without tick bites, and hounds and the other dogs but not between males and females or the group vaccinated and that unvaccinated with leptospires. According to districts, high antibody titers were observed in 20% of the dogs or more in Hokkaido, Saitama, Kanagawa, Niigata, Kyoto, Kochi, and Fukuoka Prefectures. These districts were nearly consistent with the distribution of the ticks. Western blot analysis of serum samples with a high anti-Borrelia antibody titer showed 31-, 41-, 66-, and 83-Kd bands, which are also observed in sera of human patients with LD. Though no dog had clinical signs of LD, dogs serologically positive for Borrelia were detected throughout the country. The data suggested that there were the dogs infected by Borrelia burgdorferi in Japan. The data on anti-Borrelia antibody in dogs obtained by ELISA may be very useful for studying the epidemiology of LD in humans.