Indirect immunofluorescence staining of macrophages infected with Ehrlichia risticii by anti-E. risticii serum revealed a punctate staining pattern on the surface of the host cell. This pattern was distinguishable by fluorescence microscopy from E. risticii bound to the surface of the macrophage and from intracellular E. risticii. The surface localization of ehrlichial antigen on infected macrophages was confirmed by electron microscopy with immunoferritin labeling. As the intracellular ehrlichial burden increased, the amount of ehrlichial antigen on the host cell surface increased. Prokaryotic protein synthesis was necessary for the maintenance of ehrlichial antigen on the host cell surface, as demonstrated by disappearance of the surface antigen following treatment with oxytetracycline. However, host cell protein synthesis was not required, as demonstrated by the continued presence of ehrlichial antigen on the surface of host cells after cycloheximide treatment. Pronase treatment abolished the ehrlichial antigen present on the cell surface, indicating that this antigen is a protein. Anti-E. risticii serum or immunoglobulin G-mediated antibody-dependent cellular cytotoxicity of infected cells was demonstrated in a chromium release assay. These results imply that the parasite antigen on the host cell surface has a role in the pathogenesis of ehrlichiosis.