By binding to a cis-acting element (oriP) in the Epstein-Barr virus (EBV) genome, EBV nuclear antigen 1 (EBNA1) enables persistence and enhances transcription from EBV episomes. To investigate whether EBNA1 also directly affects cell gene transcription, we conditionally expressed a Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized lymphoblastoid cell line, in which the EBV genome is integrated into cell DNA. FDNE induction inhibited expression from an EBNA1-dependent oriP reporter plasmid by more than 90% in these cells but did not affect expression from integrated EBV or oriP reporter DNA. FDNE induction also did not alter expression of more than 1,800 cellular mRNAs. Lymphoblastoid cell line growth under a variety of conditions was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly activated and coactivated a Gal4-VP16- and oriP-dependent promoter that was on an episome, only Gal4-VP16 activated the promoter when it was integrated into chromosomal DNA. These data indicate that EBNA1 is specifically deficient in activation of an integrated oriP enhancer and does not affect cell growth or gene expression through an interaction with cognate chromosomal DNA.