The ticks Amblyomma americanum and Ixodes scapularis, strongly implicated vectors of Ehrlichia chaffeensis and the human granulocytic ehrlichiosis (HGE) agent, respectively, commonly are found on white-tailed deer (Odocoileus virginianus). As deer can be infected with E. chaffeensis, the HGE agent, and another Ehrlichia-like organism, a deer population parasitized by both tick species in coastal Georgia was tested for evidence of Ehrlichia spp. infection using serologic, molecular, and culture techniques. Antibodies to both E. chaffeensis (geometric mean titer = 111) and Ehrlichia equi, surrogate antigen for the HGE agent, (geometric mean titer = 1,024) were detected by indirect fluorescent antibody testing. Nested polymerase chain reaction employing species-specific primers demonstrated sequence-confirmed 16S rDNA fragments of 3 distinct Ehrlichia spp. in this population: E. chaffeensis (1/5), the HGE agent (3/5), and an Ehrlichia-like organism previously described from white-tailed deer (5/5). Ehrlichia chaffeensis was isolated in culture from the inguinal lymph node of a single deer. An Ehrlichia-type morula was identified in a neutrophil of 1 deer on examination of blood smears. This work provides the first evidence of the HGE agent in a nonhuman host in the southeastern United States and documents infection with both E. chaffeensis and the HGE agent in a single deer population, thereby supporting the importance of white-tailed deer in the natural history of the human ehrlichioses agents.