In this study we show that macrophages (MΦ) are latently infected with murine cytomegalovirus (MCMV). After clearance of acute MCMV infection, the predominant form of chronic infection in Balb mice is latency rather than persistence. Peritoneal exudate cells (PECs) from latently infected Balb mice (3-9 months postinfection) contained MCMV genome and reactivatable virus. Adherent cells from both resident and thioglycollate-elicited PECs carried more MCMV DNA (measured by PCR) than nonadherent cells, and were selectively enriched for MΦ. FACS sorted F4/80+ MΦ contained MCMV DNA, while other FACS sorted cell populations from PECs were never positive for MCMV DNA. MCMV reactivated from FACS sorted F4/80+ MΦ in 32% of cocultures with murine embryonic fibroblasts (MEFs). Since MΦ carry MCMV genome and reactivatable virus, but not lytic virus, they are latently infected with MCMV. We determined the frequency of M carrying MCMV genome in PECs (about 1/50,000) using a limiting dilution PCR assay. Using this frequency and estimates of the total amount of MCMV genome in populations, we estimate that latently infected MΦ carry 1-10 copies of MCMV genome. To evaluate the origin of latently infected MΦ, we compared the frequency of cells carrying MCMV genome in the resident and elicited PECs. The frequency of MΦ carrying MCMV DNA was the same in resident and thioglycollate-elicited PECs, despite the fact that there was a ninefold increase in the number of MΦ recovered after thioglycollate elicitation. This argued for recruitment of bone marrow-derived MΦ (BMMΦ) carrying MCMV genome into the peritoneum during inflammatory responses. Consistent with this hypothesis, MCMV genome, but not persistent virus, was detected in bone marrow cells from latently infected mice.