Clinical experience and laboratory data suggest that human cytomegalovirus (HCMV) is present in peripheral blood of seropositive individuals in a latent or persistent state and can be transmitted via blood products and be reactivated in seropositive immunocompromised patients. The pathophysiology of HCMV latency and the nature of HCMV interaction with hematopoietic cells remains unknown. In this study, we investigated the infection of bone marrow (BM) progenitor cells and their progeny as a model of HCMV latency. A clinical isolate and the recombinant laboratory strain Towne/lox containing the Escherichia coli beta galactosidase (beta-gal) gene regulated by immediately early (IE) HCMV promoter were used to infect highly purified CD34+ cells. Although the infection of these cells with a clinical isolate was associated with an inhibition of proliferation by 59%, an expansion of progeny derived from these cells was possible. Polymerase chain reaction analysis and staining for beta-gal have shown that HCMV persisted in infected BM CD34+ cells and their progeny for up to 4 weeks. However, IE and late gene products (mRNA and protein) were detected only late in the course of infection and their expression correlated with terminal macrophage differentiation of the CD34+-derived progeny. Although early infection of CD34+ progenitor cells was not productive (as shown by the plaque assay), infectious virus could be recovered from the terminally differentiated cultures. BM progenitor cells may serve as a reservoir of the latent virus with limited transcription. Proliferation and monocytic maturation of infected progenitors may lead to the numerical expansion of HCMV-infected cells, which serve as a source of HCMV dissemination and reactivation.