It is difficult to study how Epstein-Barr virus (EBV) causes transformation of human epithelial cells. The major difficulty is that cultured human epithelial cells do not express EBV receptor (complement receptor 2, CR2), hence EBV cannot infect such epithelial cells directly. In order to investigate the role of EBV in the transformation of human epithelial cells, pSG-CR2-Hyg carrier was transfected into immortalized human epithelial cells (293 cells) to express EBV receptor. EBV could infect these CR2-positive cells directly, and expressed EBV antigens. EBV-infected epithelial cells grew in piles with multiple cellular layers and lost contact inhibition in vitro. In soft-agar culture containing 12-0-tetradecanoylphorbol 13-acetate (TPA), EBV-infected 293 cells formed more and larger colonies. When EBV-infected 293 cells were transplanted subcutaneously into nude mice, and treated with TPA, poorly differentiated carcinoma was induced. These results suggest that EBV could induce the malignant transformation of immortalized human epithelial cells in synergy with TPA.