Murine gammaherpesvirus-68 (gammaHV-68) induces a lymphocytosis in mice and establishes a latent infection of B lymphocytes following intranasal administration in anaesthetized animals. Because gammaHV-68 is a gammaherpesvirus, it has been used as a model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) infections. In this study, we investigated the unlikely possibility that gammaHV-68 could survive the harsh gastrointestinal environment to efficiently infect intestinal epithelial cells, and then disseminate from mucosal sites to cause systemic disease. Surprisingly, oral administration, or gastric instillation which by-passed the oral cavity, readily caused a systemic lymphocytosis and established a latent infection in splenic leukocytes. The finding that gammaHV-68 could readily infect adult mice following gastric instillation strongly suggested that intestinal epithelial cells could be productively infected. Unlike the more routinely used method of intranasal inoculation, gammaHV-68 given intragastrically resulted in lytic virus, viral RNA and viral DNA being present in isolated intestinal epithelial cells. Furthermore, gammaHV-68 RNA and DNA, but not latent virus, could be detected in epithelial cells as long as 30 days post-infection, suggesting that some of these cells might be persistently infected. Taken together, these studies demonstrate that gammaHV-68 can survive passage through the gastrointestinal tract and infect intestinal epithelial cells. Following infection of gut epithelial cells, gammaHV-68 can disseminate from mucosal sites to induce a systemic lymphocytosis which is similar to the disease induced following intranasal inoculation.