An infectious agent was isolated from the enlarged spleen of a wild mouse, Eothenomys kageus, by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed splenomegaly, and the agent was maintained by serial passage of spleen homogenates in laboratory mice. The agent in the spleen homogenate was inactivated after incubation at 37 or 50 degrees C. Tetracyclines were effective in preventing infection of mice with this agent, but penicillin and sulfonamides were ineffective. Cytoplasmic inclusion bodies were observed in the peritoneal macrophages of infected mice. Electron microscopy revealed numerous small pleomorphic cocci within membrane-lined vacuoles in the cytoplasm of splenic macrophages. Morphologically similar to the ehrlichial organisms, each organism was surrounded by a distinct plasma membrane and rippled outer cell membrane without a distinct peptidoglycan layer. The agent did not grow in chicken embryos, and the Weil-Felix test result was negative. In the indirect fluorescent-antibody test, the agent reciprocally cross-reacted with Ehrlichia canis and cross-reacted somewhat with Ehrlichia sennetsu but did not cross-react with Ehrlichia risticii, Neorickettsia helminthoeca, Rickettsia tsutsugamushi, or Chlamydia spp. The mouse antiserum against this agent reacted with 64-, 47-, 46-, 44-, and 40-kDa proteins of E. canis by Western blotting (immunoblotting). Since E. canis and closely related Ehrlichia chaffeensis and Ehrlichia ewingii are not known to proliferate or cause splenomegaly in mice, these results suggest that the agent is a new species within the tribe Ehrlichieae of the family Rickettsiaceae. The finding suggests that wild rodents may serve as reservoirs for pathogenic ehrlichiae.