Borna disease is a severe, immunopathological disorder of the central nervous system caused by the infection with the Borna disease virus (BDV). The detection of BDV in diseased animals, mainly sheep and horses, is achieved by histological, immunohistochemical and serological approaches and/or PCR-based technologies. In the present study, reverse transcription, real-time PCR assays were established for the detection of BDV in the brain tissue from sheep and horses, using loci for the p40 (nucleoprotein) and the p24 (phosphoprotein) genes. The PCRs were equally specific and sensitive, detecting 10 target molecules per reaction and one BDV-infected cell among 10(6) non-infected cells. In tissues from BDV-diseased sheep and horses, the p24 target was detected at higher abundance than for p40. Therefore, the p24 test is suggested to be of higher value in the diagnostic laboratory. However, both assays should be useful for addressing questions in pathogenesis and for detecting BDV reservoirs in endemic areas.