Tick infestations and infection with tick-borne agents are commonly recognized in horses in North America, but equine infection with true Ehrlichia spp. has not been described. To determine the degree to which horses in the south-central United States are naturally exposed to and infected with tick-borne disease agents, serum samples were collected at random (n=240) or from horses with active tick infestations (n=73) and tested by immunofluorescence antibody assay (IFA) and/or enzyme-linked immunosorbent assay (ELISA) for evidence of antibodies reactive to Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Positive samples were further evaluated by species-specific serology for antibodies reactive to E. canis and E. chaffeensis, and whole blood samples were tested by PCR for evidence of infection with E. canis, E. chaffeensis, E. ewingii, and an E. ruminantium-like organism referred to as the Panola Mountain Ehrlichia. Antibodies reactive to Ehrlichia spp. were identified in 8.75% (21/240) of the randomly acquired samples and 24.7% (18/73) of the serum samples from tick-infested horses, but species-specific ELISA and PCR failed to confirm exposure to or infection with any known Ehrlichia spp. Antibodies to Anaplasma spp. (5/313; 1.6%) and B. burgdorferi (3/313; 1.0%) were uncommon. These data suggest that horses in the south-central United States are likely exposed to a novel Ehrlichia sp. Further research is needed to identify the etiologic agent responsible for the serologic activity seen and to determine the clinical significance, if any, of this finding.