A rickettsia named the ELB agent, or "Rickettsia felis," was identified by molecular biology techniques in American fleas in 1990 and later in four patients from Texas and Mexico. We attempted to isolate this rickettsia from infected fleas at various temperatures and conditions. A representative isolate of the ELB agent, the Marseille strain, was characterized and used to develop a microimmunofluorescence test that detected reactive antibodies in human sera. The ELB agent was isolated from 19 of 20 groups of polymerase chain reaction-proven infected fleas. The microimmunofluorescence results provided serologic evidence of infection by the ELB agent in four patients with fever and rash in France (2) and Brazil (2), supporting the pathogenic role of this rickettsia. Our successful isolation of this rickettsia makes it available for use in serologic tests to determine its clinical spectrum, prevalence, and distribution.