To investigate the prevalence of Lyme disease infection in Taiwan, we conducted a zoonotic survey for spirochetal infection in the small mammals. Ear tissues of trapped rodents collected from various localities in Taiwan were incubated into BSK-H culture medium and examined for the evidence of spirochetal infection by dark-field microscopy. Spirochetes cultured from six species of wild and peridomestic rodents and seven isolates, designated TWKM 1-7, were purified by serial dilution and membrane filtration. Infection was detected in 16.6% (53 of 320) of captured rodents and the highest infection rate (36.4%) was observed in the brown country rat (Rattus losea, Swinhoe). Higher infection rates based on the geographic distribution were observed in the eastern localities and on Kimmen Island. Reactivity with Borrelia burgdorferi-specific monoclonal antibodies and Western blot analysis indicated that these Taiwan isolates were closely related to the causative agent of Lyme disease, B. burgdorferi sensu lato. These results provide the first evidence of the existence of Lyme disease spirochetes in the Taiwan area.