Data relating to Rickettsia typhi infection in Spain are scarce. A serological survey of 383 serum samples (184 males, 199 females) from the general population and 120 sera from sheep were studied by immunofluorescence assay (IFA). The human serum samples were collected from the general population of Palencia and Burgos provinces, and sheep serum samples were collected from Palencia province. Of the 383 human serum samples studied, 29 were positive for antibodies against R. typhi (7.5%) No statistical differences were found according to age, sex, origin (rural vs. urban) or geographic distribution, but a significant difference was observed related to occupation. In addition, 69 serums were found positives for R. typhi in sheep samples (57.5%). Our results indicated the circulation of R. typhi infectious for humans and sheep in Palencia and Burgos provinces. This study indicates that sheep may be infected with R. typhi, and that animals can, therefore be used as indicators of the presence of this organism.