Borna disease (BD) is a progressive poliomeningoencephalomyelitis which occurs naturally in horses and sheep. Here, peripheral blood mononuclear cells (PBMC) derived from 57 healthy horses in Japan were examined by a nested reverse transcription-polymerase chain reaction to determine the prevalence of BD virus (BDV) infection. Seventeen (29.8%) of the samples were positive by this examination and the specificity of the amplified product was confirmed by hybridization with authentic oligomer probes. About 60% of the BDV RNA-positive individuals also showed seropositivity by Western blotting. This report is the first for the demonstration of BDV RNA in PBMC of healthy horses, as well as the first on the BDV infection in horses in Japan. Thus, BDV may be more widespread in healthy horses over the world as well as in Japan and the detection of BDV RNA in PBMC at a high rate indicates that the disease might develop in a part of the carriers only after long-incubation period.