Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14 ؉ monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.Epstein-Barr virus (EBV) is a human herpesvirus with oncogenic potential, contributing to the development of lymphoproliferative diseases of B lymphocytes and nasopharyngeal carcinoma (18). EBV infects about 90% of the human population, but in most immunocompetent individuals EBV persists in latent form and does not cause any significant disease. During human immunodeficiency virus (HIV)-associated immunosuppression, however, EBV may reactivate and may be associated with development of a benign lesion of oral mucosal epithelium known as hairy leukoplakia (HL) (12-14). The histopathology of HL includes acanthosis, irregular hyperparakeratosis, and balloon cell formation within the spinosum and granulosum layers of the epithelium, which may result from high-level EBV replication (14).HL is a common lesion in HIV-positive patients with low CD4 ϩ counts, suggesting that immunosuppression is an important factor in its development. The source of the EBV in HL is not known. Several lines of evidence support hematogenous spread from circulating white blood cells (WBC) (11,29). The main reservoir of latent EBV infection in the body is memory B lymphocytes, but mechanisms of spread from cells in the blood compartment to the mucosal epithelium are not known. Furthermore, HL epithelium may support both latent and lytic replication of EBV (43), with lytic EBV replication and cellto-cell spread of virions restricted exclusively to the terminally differentiated stratum spinosum and granulosum layers (28,31,44). Neither the mechanisms by which EBV enters ...