Epstein-Barr virus (EBV) prevalence was assessed in 12 clinically normal oral mucosas, nine premalignant lesions, 36 oral squamous cell carcinomas (OSCCs) and a human papillomavirus (HPV) 16 positive cell line, derived from an OSCC studied. The polymerase chain reaction (PCR) with two pairs of primers with different sensitivities was used. With primers specific for the BamHIW repeat, EBV was found in 100% of OSCCs, in 77.8% of premalignant lesions and in 8.3% of clinically normal oral mucosas (P = 0.0001). Using primers specific for the single copy BNLF-1 gene, EBV was detected in 50% of OSCC and in none of the premalignant lesions. No statistically significant associations could be established among EBV presence and clinico-pathological data of OSCC. The cell line derived from an HPV/EBV-positive carcinoma did not reveal EBV DNA. The higher prevalence of EBV in OSCCs and premalignant lesions may be due to increased EBV shedding, possibly due to associated immunodepression in these patients, rather than its clonal presence in the neoplastic cells.