White-tailed deer (Odocoileus virginianus) play an integral role in the natural history of Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME). Paraffinized tissues from a white-tailed deer submitted as a diagnostic case to the Southeastern Cooperative Wildlife Disease Study (Athens, Georgia, USA) in October of 198.5 and originally described as infected with an unidentified rickettsial organisim were re-examined by specific nested polymerase chain reaction (PCR) for evidence of infection with Ehrlichia spp. Ehrlichia chaffeensis was identified from the bone marrow and inguinal lymph node of this deer based on amplification of a characteristic sequence-confirmed 16S rDNA fragment from these tissues. Parallel PCR tests on the same samples were negative for 16S rDNA fragments of the agent of human granulocytic ehrlichiosis (HGE) and for an Ehrlichia-like organism widely distributed in white-tailed deer populations. This report describes detection of E. chaffeensis in archived tissue from a deer collected before the index case of human monocytic ehrlichiosis was established.