It is currently believed that latently infected, resting B lymphocytes are central to gammaherpesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have readdressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV-68). To dissect lymphoid from nonlymphoid persistence, we used microMT transgenic mice that are defective in B cells. MHV-68 DNA persisted in the lungs of intact and B cell-deficient mice. Both episomal and linear forms of the virus genome were present in lungs, implying the presence of both latency and productive replication. In situ hybridization for virus tRNA transcripts revealed latent MHV-68 in pulmonary epithelial cells. Infectious virus was recovered from the lungs of microMT mice after T cell depletion, showing that the persisting virus DNA was reactivatable. Finally, using adoptive transfer of B cells into B cell-deficient mice, it was shown that virus persisting in lungs seeded splenic B cells, and virus resident in the spleen seeded the lungs. These results show that mucosal epithelia can act as a nonlymphoid reservoir for gammaherpesvirus persistence, and that there is a two-way movement of virus between lymphoid and nonlymphoid compartments during persistence.