Reactivation of human cytomegalovirus (HCMV) results in severe disease in AIDS patients and immunocompromised patients receiving blood transfusions or organ or bone marrow grafts. Although the site of HCMV latency is unknown, blood cells have been implicated as a viral reservoir. In this study, we demonstrate HCMV reactivation in vitro from seven consecutive healthy donors through allogeneic stimulation of peripheral blood mononuclear cells (PBMCs). HCMV replication was detected at 17 days poststimulation, and virus was recovered after long-term culture from a macrophage expressing dendritic cell markers. Thus, these observations demonstrate that PBMCs harbor latent HCMV, which reactivates in a myeloid lineage cell upon allogeneic stimulation.