Two rickettsial isolates recovered from Ixodes ricinus ticks in Puy-de-Dôme (Central France) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot immunoassay, restriction fragment length polymorphism analysis, and sequencing of a portion of the citrate-synthase gene. By these methods, the isolates appeared to be identical to a member of the spotted fever group rickettsiae, Rickettsia helvetica. This first isolation of Rickettsia helvetica in France has epidemiologic importance; a serosurvey on Mediterranean spotted fever conducted previously in Puy-de-Dôme, where the infection is not endemic, demonstrated a high seroprevalence of nonspecific antibodies directed against spotted fever group rickettsiae lipopolysaccharides, and thus the possibility of infection due to a rickettsia different from Rickettsia conorii was suggested. The isolation of Rickettsia helvetica in anthropophilic ticks in the same area further supports this hypothesis.