Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.