The hairy leukoplakia lesion (HLP) is a unique example of a permissive infection with Epstein-Barr virus (EBV) in the tongue epithelium. HLP contains abundant replicating viral DNA and may be coinfected with multiple EBV strains. In this study, characterization of viral gene transcription within HLP biopsy specimens revealed that several genes, usually expressed in latently infected lymphocytes, are also transcribed in the HLP lesion. The BamHI W and C promoters, (Wp and Cp) are consistently active in the HLP lesion, resulting in transcription and processing of mRNAs that encode the Epstein-Barr nuclear antigens (EBNAs) EBNA-LP, EBNA1, EBNA2, EBNA3B, and EBNA3C. The EBNA2 protein has been shown to activate expression of the EBV receptor, CD21. In HLP, CD21 transcription is also detected, usually in samples that contain transcripts for EBNA2. Transcripts encoding the LMP1 gene, the LMP2 gene, and rightward transcripts from the BamHI A fragment of the EBV genome are also detected in HLP. These gene products are invariably expressed in latently infected lymphocytes. This pattern of transcription suggests that genes characteristic of latent infection are also expressed in HLP. The activation of Wp and expression of EBNA2 and CD21 may contribute to the unique ability of the HLP lesion to permit superinfection and viral replication of multiple EBV strains.