An Ehrlichia sp. (Panola Mountain [PM] Ehrlichia sp.) closely related to Ehrlichia ruminantium was recently detected in a domestic goat experimentally infested with lone star ticks (LSTs, Amblyomma americanum) collected from Georgia, USA. The infected goat exhibited pyrexia and mild clinical pathologic abnormalities consistent with ehrlichiosis. At least two other Ehrlichia species (Ehrlichia chaffeensis and Ehrlichia ewingii) are maintained in nature by a cycle involving LSTs as the primary vector and white-tailed deer (Odocoileus virginanus) as a known or suspected reservoir. To investigate the possibility that white-tailed deer are potential hosts of the PM Ehrlichia sp., whole blood samples collected from 87 wild deer from 2000 to 2002 were screened with a species-specific nested PCR assay targeting the citrate synthase gene. In addition, two laboratory-raised white-tailed deer fawns were each infested with 120 wild-caught LST adults from Missouri, USA, and blood samples were periodically collected and tested for the PM Ehrlichia sp. Of 87 deer tested from 20 locations in the southeastern United States, three (3%) deer from Arkansas, North Carolina, and Virginia were positive for the PM Ehrlichia sp. Wild-caught ticks transmitted the PM Ehrlichia sp. to one of two deer fawns, and colony-reared nymphal LSTs acquired the organism from the deer, maintained it transstadially as they molted to adults, and transmitted the PM Ehrlichia sp. to two naïve fawns. These findings indicate that white-tailed deer are naturally and experimentally susceptible to infection with an Ehrlichia sp. closely related to E. ruminantium and are able to serve as a source of infection to LSTs.