Ehrlichia chaffeensis, etiologic agent of human ehrlichiosis, and Rickettsia rickettsii, etiologic agent of Rocky Mountain spotted fever (RMSF), are both tick-borne agents that cause nonspecific symptoms that may be indistinguishable from each other early in the course of infection. E. canis is a canine pathogen closely related to E. chaffeensis and was initially suspected of being the causative agent of human ehrlichiosis. If a febrile illness is reported, after tick exposure, neither ehrlichiosis nor RMSF can be immediately ruled out. When attempts are made to isolate the agent from blood, a very limited amount of blood is often available; we, therefore, sought a tissue-culture cell line that would support the growth of both R. rickettsii and E. chaffeensis. A newly established human microvascular endothelial immortal cell line (CDC/EU.HMEC-1) was evaluated for supporting the growth of both agents. Our results demonstrate that HMEC-1 supports the growth of R. rickettsii, E. chaffeensis, and E. canis and may be a useful tool for the isolation of these agents.