Background and Aims: Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas; however, localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the present study, we measured EBV DNA and localized viral gene products in gastritis specimens (n = 89), normal gastric and colonic mucosa (n = 14), Crohn's disease (n = 9), and ulcerative colitis (n = 11) tissues. Methods A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1 W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains. Results: EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn's disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR. Conclusion EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation. © Springer Science+Business Media, LLC 2012.