Lyme disease, caused by the spirochete Borrelia burgdorferi, is endemic in the northeast, north-central, and Pacific coastal states of the United States. The eastern chipmunk (Tamias striatus) is found throughout the disease-enzootic regions except along the Pacific coast, and may serve as an important reservoir host in some locations. To characterize their potential as a host, 11 adult chipmunks were inoculated with 10⁵ spirochetes from strains of B. burgdorferi isolated from Peromyscus leucopus and Ixodes scapularis in a hyperendemic area of Westchester County, New York (USA). All inoculated chipmunks became infected. Spirochetemias were detected by isolating spirochetes in Barbour-Stoenner-Kelly media in eight of eight chipmunks, and lasted for 2 to 5 days. Spirochetes were isolated from the ears of all animals, starting at 1 wk and for ≤4 mo, and from various internal organs at 133 days post-inoculation. Laboratory-reared larval I. scapularis ticks became infected with spirochetes after feeding on two of the inoculated chipmunks.