Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia. Epstein-Barr virus (EBV) DNA structure and gene expression were analyzed in tissue specimens from oral hairy leukoplakia (HLP), a mucocutaneous lesion that develops in patients infected with human immunodeficiency virus (HIV). The structure of the terminal restriction enzyme fragments of EBV revealed that HLP is a permissive infection without a predominant, detectable population of EBV episomal DNA. In RNA preparations from this uniquely permissive infection, EBV replicative mRNAs could be identified by Northern analysis; however, the virally encoded small nuclear RNAs, the EBERs, were not detected in most HLP RNA preparations. In situ hybridization detected EBER expression in very rare cells. These data indicate that unlike other viral small nuclear RNAs, the EBERs are not expressed during viral replication and must participate in the complex maintenance of latent EBV infection. 