Tick-borne pathogens in the genus Ehrlichia cause emerging zoonoses. Although laboratory mice are susceptible to Ehrlichia infections, many isolates do not cause clinical illness. In contrast, the Ixodes ovatus Ehrlichia-like agent (IOE) causes disease and immune responses in mice comparable to the human illness caused by Ehrlichia chaffeensis. No culture system had been developed for IOE, however, which limited studies of this pathogen. We reasoned that endothelial and tick cell lines could potentially serve as host cells, since the IOE is found in ticks and in endothelial cells in mice. Infected spleen cells from RAG-deficient mice were overlaid onto ISE6 and RF6A cultures, and colonies typical of Ehrlichia were noted in RF6A cells within 2 weeks. Infection of ISE6 cells was established after transfer of IOE from RF6A cells. Electron microscopy revealed densely packed inclusions in infected RF6A and ISE6 cells; these inclusions contained copious amounts of filamentous structures, apparently originating from Ehrlichial cells. In particular, within RF6A cells the structures assumed an ordered morphology of finely combed hair. IOE from RF6A cells, when inoculated into C57BL6 and RAG-deficient mice, induced fatal disease. These data reveal unique structural features of IOE that may contribute to the pathogen's high virulence.