Periodontitis is presumably caused by bacterial infection, but it has been shown recently that affected tissue often contains human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The present study was initiated to evaluate the role of these viruses in the pathogenesis of periodontitis. HCMV and EBV were quantified in 40 apical and 25 marginal periodontitis samples using real time PCR. In situ hybridization or immunohistochemistry was carried out on apical samples to detect viral presence within cells. A possible association with relevant bacteria was examined. Of the apical periodontitis samples, 50% contained EBV, while none contained HCMV. Of the marginal periodontitis samples, 40% were positive for EBV and 12% for HCMV. With one exception, however, the amount of virus was close to the detection limits. EBV was only detected in 1 out of 15 healthy periodontium samples. Immunohistochemistry and in situ hybridization were all negative. Significant associations were found between periodontal EBV and the presence of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Although there was an obvious association of the virus with clinical samples, it seems unlikely that these viruses play a major role in the pathogenesis of periodontitis of the average patient. Their presence may reflect that the clinical samples contain more blood or saliva compared to controls, or an accumulation of lymphoid cells harboring virus in the inflamed tissue.