The disease bovine besnoitiosis is responsible for severe economic losses caused by the protozoan Besnoitia besnoiti. The identity of the definitive host (DH) of this parasite has yet to be determined, although it is presumed to be a carnivore. With the aim of advancing in the identification of B. besnoiti DH, a necessary step in implementing control strategies, the contact rate of 205 free-roaming carnivores with this parasite in Spain was studied. The study included 16 wolves (Canis lupus), 41 red foxes (Vulpes vulpes), 21 pine martens (Martes martes), eight stone martens (M. foina), 12 Eurasian badgers (Meles meles), 18 common genets (Genetta genetta), five Egyptian mongooses (Herpestes ichneumon), 28 European wildcats (Felis silvestris silvestris), 43 feral cats (Felis silvestris catus), and 13 other animals belonging to five other species. Serum samples were analysed by an indirect fluorescent antibody test (IFAT) and by two western immunoblots (WB, one with tachyzoite and the other with bradyzoite antigen). Twelve individuals (eight of which were cats) seroconverted by one or other of these techniques but no individual showed seroconversion by IFAT and one of the WBs. The results provided no evidence to support the idea that within the geographical regions covered by the analysis wild carnivores are implicated in the transmission of B. besnoiti in Spain. © 2012 Elsevier B.V.