Serum samples from white-tailed deer, Odocoileus virginianus Zimmermann, collected from 1982 through 1992 from the southeastern United States were tested for antibodies reactive to Ehrlichia chaffeensis Anderson, Dawson, Jones, & Wilson, the causative agent of human ehrlichiosis. Results were compared between areas based on known infestations of the lone star tick, Amblyomma americanum L., a suspected vector of E. chaffeensis. One hundred and twenty-five of 300 (41.7%) deer tested positive (> or = 1:128) for E. chaffeensis-reactive antibodies by fluorescent antibody analysis. Thirty of 30 (100%) collection areas known to be lone star tick infested contained deer that tested positive for E. chaffeensis-reactive antibodies, corresponding to 121/150 (80.7%) of deer examined. A few deer, 4/150 (2.7%) of those examined, from 2 of 30 (6.7%) areas where lone star ticks were not detected were positive for E. chaffeensis-reactive antibodies. This site-specific geographic association between A. americanum and the presence of E. chaffeensis-reactive antibodies in deer provides strong evidence that A. americanum is a natural vector of E. chaffeensis or a closely related species among white-tailed deer.