It is known that the HER2/neu proto-oncogene is associated with a wide variety of human cancers and considered to be an attractive target for developing anti-cancer agents. We report here for the first time that the Epstein-Barr virus nuclear antigen-1 (EBNA1) suppresses the HER2/neu oncogene expression at the transcriptional level. Recombinant clones of EBNA1 were subcloned and stably transfected into HER2/neu-overexpressing human ovarian cancer SKOV3.ip1 cells. These EBNA1-containing clones down-regulated the endogenous production of p185(HER2/neu). In addition, the EBNA1-expressing stable transfectants showed reduced growth rate, low soft agarose colony-forming ability and tumorigenic potential as compared with the parental line. These data suggest that EBNA1 may act as a transforming suppressor of the HER2/neu oncogene.