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Macrophages (Mφ) harvested from the peritoneal cavities of mice after thioglycollate stimulation could be infected with murine cytomegalovirus (MCMV), although the efficiency of infection was low. Sequential measurements of interferon (IFN) production by virus-infected Mφ were performed in an attempt to explain the characteristics of MCMV infection in the cell cultures. Infected Mφ produced moderate amounts of IFN, which was completely neutralized by anti-IFN-α/β serum. The IFN was detectable in cultures as early as 8 h after infection and was produced only by exposing Mφ to infectious virus. Production increased until 48 to 72 h and preceded virus production, which was initially detected 72 h after infection. Treatment of the Mφ cultures with anti-IFN-α/β resulted not only in a marked increase in virus production, as well as a shortening of the long eclipse period of MCMV infection, but also induced increases in the number of Mφ releasing MCMV (VR-Mφ). Thus, the IFN produced in MCMV-infected Mφ (MCMV-Mφ IFN) appeared to suppress the production and spread of MCMV. The increase in the number of VR-Mφ observed was more resistant to anti-IFN-α/β treatment than the production of infectious virus. The antiviral effect of MCMV-Mφ IFN on MCMV infection in mouse embryo fibroblasts was similar to that induced by IFN-α/β. Therefore, MCMV-Mφ IFN appeared to be more active in protecting against the spread of cell-free MCMV than of cell-associated virus. These differences in sensitivity to IFN action suggest that Mφ may have a role in the latency of MCMV and that their production of IFN may facilitate the generation of latent infection.