The definitive diagnosis of oral hairy leukoplakia (OHL) demands that Epstein-Barr virus (EBV) is demonstrated in the lesional tissue, since the histopathological features on conventional light microscopy are not pathognomonic. We have investigated the possible use of polymerase chain reaction (PCR) technology in reaching a definitive diagnosis of this lesion by its application to ten biopsy specimens with definitive diagnoses of OHL determined by in situ hybridization. EBV DNA was demonstrated by PCR in all ten OHL biopsy specimens analysed, and none of ten control specimens. Furthermore, we have investigated the role of PCR in analysis of exfoliative cytology samples collected from the lateral border of the tongue by a minimally-invasive scraping technique. EBV DNA was not only detected in all OHL lesional scrapings but also in more than one-third of healthy controls, due to viral presence in saliva at the time of sampling. In this application, the highly sensitive PCR technique has low specificity and cannot be recommended.