Non-neoplastic tonsils were analyzed for detection of Epstein-Barr virus (EBV)-positive cells by in situ hybridization and in situ PCR. EBV-encoded small nuclear RNA 1(EBER1)-positive cells were found in 28.2% of the tonsils and were evenly localized in the extrafollicular area and within germinal centers. Latent membrane protein 1 (LMP1)-positive cells were also dispersed in the extrafollicular and germinal center. Using in situ DNA-DNA hybridization, the EBV-positive signals were observed in the upper epithelial cell layers of the tonsils. In addition, in situ PCR detected EBV DNA-positive cells in the lower epithelial cell layers and lymphoid cells.