To determine whether cats could be infected experimentally with Borrelia burgdorferi, 15 cats were inoculated with approximately 1,000 B burgdorferi. Seven cats were inoculated by the IV route, 2 by the oral route, 2 by the ocular route, and 4 by the oral-ocular route. Six control cats were inoculated with phosphate-buffered saline solution by the IV, oral, and ocular routes. Prior to the start of the study, all 21 cats were seronegative for B burgdorferi on the basis of results of the indirect fluorescent antibody (IFA) test, and their blood was B burgdorferi culture negative. All of the IV, orally, and ocularly inoculated cats developed IgG antibodies to B burgdorferi as detected by IFA testing. Of 4 oral-ocularly inoculated cats, 2 developed IFA-detectable antibodies and the remaining 2 cats developed low-titer response (1:128) on postinoculation (PI) day 10 only. All control cats remained seronegative. The organism was detected in blood smears from 2 of the IV inoculated cats on PI days 10 and 24 and from 2 oral-ocularly infected cats, 1 on PI days 17 and 24 and 1 on PI day 10. Spirochetes were not detected in the blood after PI day 24. The organism was isolated from tissues of only 1 cat (the lung of an ocularly inoculated cat necropsied at 7 months after inoculation). Spirochetes were not isolated from control cats. Neither clinical signs of infection nor gross or histologic abnormalities were found in any of the inoculated or control cats. Results indicate that cats are susceptible to infection with B burgdorferi, but clinically apparent disease may not be common.