The close association of Epstein-Barr virus (EBV) and nasopharyngeal carcinoma (NPC) is well documented and the expression of EBV-related latent genes has been shown in NPC. Nevertheless, the status of EBV infection in the nontumor epithelial and lymphoid cells is not known. In this study, we detected EBV in the nontumor component of NPC and nontumor nasopharyngeal biopsies by in situ hybridization using digoxigenin-labeled antisense EBER1 oligoprobe in 346 nasopharyngeal biopsies. Latent membrane protein (LMP) and ZEBRA were detected by immunohistochemistry in cases containing EBER1-positive cells. The EBV-positive epithelial and lymphoid cells were identified by immunostains for cytokeratin and lymphoid phenotypes. In the nontumor nasopharyngeal biopsies, 21 (11.7%) of 179 cases had EBV-harboring lymphocytes in the lymphoid tissue, while the overlying mucosa was all negative. LMP was demonstrated in lymphoid cells of 14 (66.7%) of these 21 samples. These EBV-positive lymphocytes were B lymphocytes by combined phenotype study. EBER1-containing metaplastic squamous cells were demonstrated in the overlying mucosa close to the tumor tissue in 14 (8.4%) of 167 NPC samples. In contrast, the respiratory epithelial cells in these cases were all negative. Two (1.2%) of these cases had EBV-positive lymphocytes in the lymphoid stroma. Four of the 346 cases had ZEBRA-positive cells. This study demonstrated that latent infection and, occasionally, active replication of EBV were present in stromal B lymphocytes in nasopharyngeal tissue with and without NPC. EBER1 was found only in the metaplastic squamous cells in the mucosa of nasopharyngeal tissues with NPC, but not in those without NPC.