All currently available assays for antibodies to the etiologic agents of spotted fever group rickettsioses detect reactivity with antigens of lipopolysaccharides and the major cell wall proteins, which contain epitopes that are shared among many spotted fever group rickettsiae. The hypothesis of this study was that a monoclonal antibody to a species-specific epitope of Rickettsia rickettsii would be blocked from binding to the rickettsial surface if the rickettsiae were incubated previously with serum containing species-specific antibodies to the epitope. In an EIA, binding of monoclonal antibody 5C10-F3 to R. rickettsii was blocked by convalescent sera from patients with Rocky Mountain spotted fever but not from those with Mediterranean spotted fever, endemic typhus, or Q fever or normal subjects. This assay should be useful in determining the origin of the high seroprevalence of spotted fever rickettsial antibodies in certain geographic regions and establishing a species-specific diagnosis in patients with undetermined rickettsial exposure.