Macrophages (M phi) 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 M phi were performed in an attempt to explain the characteristics of MCMV infection in the cell cultures. Infected M phi produced moderate amounts of IFN, which was completely neutralized by anti-IFN-alpha/beta serum. The IFN was detectable in cultures as early as 8 h after infection and was produced only by exposing M phi 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 M phi cultures with anti-IFN-alpha/beta 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 M phi releasing MCMV (VR-M phi). Thus, the IFN produced in MCMV-infected M phi (MCMV-M phi IFN) appeared to suppress the production and spread of MCMV. The increase in the number of VR-M phi observed was more resistant to anti-IFN-alpha/beta treatment than the production of infectious virus. The antiviral effect of MCMV-M phi IFN on MCMV infection in mouse embryo fibroblasts was similar to that induced by IFN-alpha/beta. Therefore, MCMV-M phi 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 M phi may have a role in the latency of MCMV and that their production of IFN may facilitate the generation of latent infection.