Herpesviruses typically establish latent infection in their hosts. The cell(s) responsible for harboring latent virus, in most cases, is not known. Using immunofluorescence and PCR-in situ hybridization (PISH), a technique which combines the sensitivity of PCR with the localization and specificity of in situ hybridization, we provide the first direct evidence that endothelial cells are a major site of murine cytomegalovirus (MCMV) DNA in latently infected animals. These findings are consistent with existing knowledge of the biological behavior of CMV, in particular the transmission of latent CMV by solid organ and bone marrow transplantation, in both human and animal models. In addition, we have localized MCMV DNA in the lung alveolar macrophage and in bone marrow cells. Our findings confirm that bone marrow-derived hematopoietic cells are a site of CMV latency and further suggest that bone marrow may be a reservoir of infected progeny capable of migrating into the circulation and establishing latency in various tissues. These findings provide clearly needed insight into the site of latent infection which is central to an understanding of the mechanisms of reactivation.