Rickettsia helvetica, a tick-borne member of the spotted-fever-group rickettsiae, is a suspected pathogen in humans; however, its role in animals is unknown. The aims of this study were to establish a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (to determine potential exposure risk) and blood samples from Canidae and humans (to determine prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase gene (gltA) assay for several rickettsiae. Blood samples from 884 dogs, 58 foxes, and 214 human patients and 2,073 ticks (Ixodes spp.) collected from either vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR positive. Ticks from cats were more frequently PCR positive than ticks from dogs. Sequencing of the 23S rRNA and/or the gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, Rickettsia monacensis, which has not been previously found in Switzerland, was identified. In conclusion, R. helvetica was frequently detected in the tick population but not in blood samples. Nevertheless, due to the broad host range of Ixodes ticks and the high rate of infestation with this agent (i.e., R. helvetica was 13 times more frequent in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The PCR assay described here represents an important tool for studying this topic.Tick-borne rickettsioses are caused by intracellular bacteria belonging to the spotted fever group (SFG) of the genus Rickettsia. The latter comprises more than 20 different species, of which an increasing number are known to be associated with human and animal diseases. The SFG rickettsiae are distributed worldwide, and their distribution depends upon the occurrence of tick species. The most common tick in Europe is Ixodes ricinus, which was found to harbor Rickettsia helvetica. R. helvetica is transmitted not only transstadially but also transovarially in I. ricinus. Therefore, this tick is both a vector and a reservoir for R. helvetica. Due to the broad host range of I. ricinus, many mammalian species, including humans, can serve as hosts. Therefore, these host species may potentially be exposed to R. helvetica. R. helvetica is a suspected pathogen in humans, and the symptoms described for infections in humans include fever, headache, arthralgia, and myalgia (1, 3, 7, 21, 34). The agent also has been implicated in two cases of fatal perimyocarditis (20,22).Interestingly, despite the wide distribution of I. ricinus ticks and the high rate of infection of these ticks with R. helvetica that has been reported in several European countries (2,9,18,19,25,29,35,42), larger studies discussing the prevalence of the infection in humans and animals are scarce. No studies evaluating the importance of R. helvetica in pets or farm animals are ...