The role of white-tailed deer (Odocoileus virginianus) in the epidemiology of Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (HGE) is not fully understood, and diagnostic procedures may be complicated by the recent detection of 16S rDNA sequence from an Ehrlichia sp.-like organism in wild deer. A specific forward primer (DGA) and an Ehrlichia spp. reverse primer (GA1UR) were constructed to amplify this new, distinct Ehrlichia sp.-like 16S rDNA. The DGA primer, a forward primer specific for E. chaffeensis (DCH), and a forward primer specific for the E. phagocytophila genogroup (GE9f) were each used with GA1UR in nested polymerase chain reactions to amplify 16S rDNA sequences from control samples containing the deer Ehrlichia sp.-like organism, E. chaffeensis, or the HGE agent. Primer pairs DGA/GA1UR and DCH/GA1UR specifically amplified 16S rDNA sequences from the corresponding target organism, whereas GE9f/GA1UR amplified 16S rDNA sequence from both the HGE agent and the deer Ehrlichia sp.-like organism. With a nested PCR using DGA/GA1UR and DCH/GA1UR on DNA extracted from white blood cells from 62 deer from 10 populations in four U.S. states, we observed a high prevalence (65%) of 16S rDNA sequences of the deer Ehrlichia sp.-like organism, and a low prevalence (5%) of the E. chaffeensis sequence. In this field survey, E. chaffeensis-reactive antibodies detected by indirect fluorescence assays were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism, but not E. chaffeensis. Infestations of Amblyomma americanum also were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism. The potential for serologic cross-reactions and non-specific PCR products arising from the deer Ehrlichia sp.-like organism should be considered when evaluating the role of deer and their ticks in the epidemiology of ehrlichial pathogens of humans.