Although more than 320 cases of human ehrlichiosis have been diagnosed in 27 states since 1986, the reservoir host or hosts remain unknown. Since antibodies reactive to Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis, have been found in white-tailed deer (Odocoileus virginianus), we experimentally evaluated the susceptibilities of four white-tailed deer to infection with E. chaffeensis and Ehrlichia canis, a closely related species. A fifth deer served as a negative control. Isolation and nested PCR amplification results from peripheral blood indicated that E. chaffeensis circulated for at least 2 weeks. The deer developed antibodies to E. chaffeensis by day 10 after inoculation, but there was no indication of clinical disease. Immunohistochemical staining identified E. chaffeensis within macrophage-type cells in lymph nodes. The deer inoculated with E. canis did not become infected and did not seroconvert. These results indicate that white-tailed deer can support an E. chaffeensis infection with resulting rickettsemia of at least 2 weeks. The resistance to infection and the absence of seroconversion upon exposure to E. canis indicate that antibody responses previously detected among wild deer are not E. canis cross-reactions. The role of deer as competent reservoirs in the life cycle of E. chaffeensis remains to be explored with suspected tick vectors.