Epstein-Barr virus (EBV) infects both B lymphocytes and oropharyngeal epithelium, and it has been argued that the true reservoir of virus persistence in vivo is the self-renewing basal epithelial compartment. The identification of oral hairy leukoplakia (HL) of AIDS patients as a clinically apparent focus of EBV replication in lingual epithelium therefore provides a means of studying the EBV-epithelial cell interaction in situ. Replicative EBV DNA and productive cycle antigens are restricted to the upper, more differentiated epithelial layers in HL, and here we have applied highly sensitive in situ hybridization and immunohistological methods to examine the lower basal/suprabasal layers for evidence of latent EBV infection. We could not detect EBV DNA in these layers using an in situ DNA hybridization protocol which, on reference B cell lines, detected 1 viral genome/cell. Likewise, using sensitive in situ RNA hybridization for both the small non-polyadenylated EBER RNAs (abundant transcripts seen in all known forms of EBV latency) and the latent membrane protein (LMP) mRNA (the most abundant viral mRNA in B lymphoblastoid cell lines), the basal/suprabasal cells in HL were consistently negative; immunohistological staining with specific monoclonal antibodies also gave no evidence of latently infected LMP-positive cells. When the biopsy extracts were analysed by immunoblotting with selected human antisera, in addition to abundant productive cycle antigens, a band of constant size (66K) was observed which also reacted with immunopurified antibodies monospecific for one of the latency-associated nuclear antigens, EBNA 1; the cellular origin of this EBNA 1 could not be ascertained, but it is possible that in HL the protein is expressed during the productive cycle. The absence of demonstrable EBV latency in the basal/suprabasal cells of HL suggests that this is purely a virus replicative lesion which is sustained by continual re-infection of the maturing epithelium, not by the maturation of latently infected cells from the basal compartment.