Borna disease virus (BoDV) can cause neurological disease in several vertebrate species, especially in horses and sheep. The recent cases of fatal encephalitis caused by BoDV in humans have raised concern regarding the zoonotic character of this virus. BoDV infection in horses is endemic in Central Europe, whereas in Spain the disease is regarded as an exotic disease in horses and serological investigations in these animals have never been performed. The aim of this study was to investigate the exposure to BoDV in breeding and sport horses in Spain and to determine risk factors for the transmission of the virus. Serum samples from 495 horses (260 sport horses and 235 Spanish Purebred breeding horses) were tested by an Indirect Fluorescence Antibody Test (IFAT). From the SP breeding horses we examined factors putatively associated with seropositivity to BoDV. A total of 164 horses (33.1%; 95% CI 29.0-37.3) showed antibodies against BoDV. Factors associated with BoDV seropositivity in the SP breeding horses were climate, age and the time of the year when the horses were sampled. This is the first report on the presence of BoDV antibodies in apparently asymptomatic breeding and sport horses in Spain. Our results demonstrate the need for BoDV surveillance in horses and possible reservoir hosts in this country, and suggests that BoDV should be included in the differential diagnosis of equine and human cases of encephalitis.