Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever (MSF). A new procedure using indirect immunofluorescence was used to directly detect R. conorii in circulating endothelial cells (CEC). CEC were separated from other blood components by using anti-endothelial cell monoclonal antibody-coated magnetic beads. An anti-R. conorii polyclonal rabbit antiserum was used to stain rickettsiae. The entire procedure took 3 h. R. conorii was detected in CEC from 9 of 12 patients ultimately confirmed as having MSF. Among the patients, 5 with R. conorii isolated by centrifugation-shell vial assay were also positive by the new technique. None of 3 patients whose diagnosis was other than MSF had R. conorii detected in CEC. The procedure may be helpful for rapid diagnosis of MSF and may lead to new technical approaches for the diagnosis of infectious diseases caused by intracellular pathogenic microorganisms.