We have investigated the interaction of human cytomegalovirus (CMV) with cultured primary granulocyte-macrophage progenitors, a suspected natural site of viral latency, and have established conditions for latent infection and reactivation in this cell population. Progenitor cells from human fetal liver or bone marrow maintained a CD14+, CD15+, CD33+ cell surface phenotype during propagation in suspension culture. Exposure to human CMV did not reduce growth or alter the phenotype of these cells during a 4-week culture period. Viral replication was not detectable in these cells, although viral DNA, as measured by PCR analysis, persisted in a high proportion of cultured cells in the absence of delayed early (beta) gene expression. Viral gene expression was restricted such that only ie1 region transcripts were detected by PCR analysis of cDNA, and these transcripts were estimated to be present in no less than 2-5% of latently infected cells. Most of these transcripts remained unspliced, a result that strikingly contrasts with the splicing pattern normally seen during viral replication in permissive cells. Latent virus reactivated after prolonged, 16- to 21-day cocultivation of infected granulocyte-macrophage progenitors with permissive cells, results that support a role for the myelomonocytic cell population as a biological reservoir of latent human CMV and suggest that these cells may be the source of CMV DNA PCR-positive monocytes found in the peripheral blood of healthy carriers.