The aim of this study was to investigate Epstein-Barr virus (EBV)-related virus infection in pet dogs. The presence of antibodies to EBV antigens and EBV-related DNA was determined by Western blot analysis and PCR, respectively. Among 36 pet dogs examined for serum antibodies, 32 (88.9%) were positive for EBV-specific thymidine kinase, 15 (41.7%) for EBV-encoded DNA-binding protein and 10 (27.8%) for EBV-specific DNA polymerase. A BamHI W fragment sequence encoding part of the EBV nuclear antigen leader protein was detected by PCR in corresponding leukocyte DNA samples. Among 21 dogs tested, 15 (71.4%) were positive for the BamHI W fragment sequence. The specificity of the amplified DNA fragments was confirmed by DNA sequencing. Within the amplified region of the BamHI W fragment (241 bp), DNA sequences detected in 10 dogs had 99.2% (two nucleotide variations), 99.6% (one nucleotide variation) or 100% identity to that of EBV. Furthermore, an EBV-encoded RNA signal was detected by in situ hybridization in dog lymphocytes, as well as in bone-marrow sections, indicating a latent infection with EBV or an EBV-like virus. In conclusion, although the sample size was small, these results showed that a widespread EBV-related gammaherpesvirus could be detected in the peripheral blood and bone marrow of pet dogs. Although no evident zoonotic transmission was detected, further studies are imperative for disclosing the biological significance of this canine EBV-like virus, which may correlate with human disorders.