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NPC-KT cl.S61, a subclone derived from an epithelial-nasopharyngeal carcinoma hybrid cell line NPC-KT, showed extensive Epstein-Barr virus (EBV) production and cell fusion when the EBV replicative cycle was induced by 5-iodo-2‘-deoxyuridine. On the contrary, parental NPC-KT cells produced virus at a lower level and did not show cell fusion. Cell fusion in cl.S61 cells was blocked by 2-deoxyglucose and acyclovir, inhibitors of glycosylation and EBV DNA polymerase, respectively, with a concomitant decrease in the number of cells expressing EBV growth-associated antigens. However, the frequency of virus antigen expression in parental NPC-KT cells was not significantly affected by these drugs. This result suggests that efficient production of EBV from cl.S61 cells is due to the spreading of viral replicative cycle via cell fusion. It was also demonstrated by in situ autoradiography that cl.S61 cells producing virus fused to not only EBV receptor/CR2 positive Raji and BJAB cells, but also to receptor-negative Jurkat cells. The possible mechanism of EBV entry into cells devoid of virus receptor by cell fusion is discussed.