Vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) multiply in only a small percentage of peritoneal macrophages freshly explanted from 4- to 6-week-old male or female DBA/2, BALB/c, C3H, C57BL/6, or Swiss mice. However, when these mice were injected intraperitoneally with potent sheep (or goat) anti-mouse interferon alpha/beta globulin 4 days prior to harvesting peritoneal macrophages, the viruses multiplied to high titers and most of the cells were infected, as determined by total virus yield (VSV and EMCV), percentage of VSV antigen-positive cells (immunofluorescence), and determination of VSV infectious centers. This effect was not observed when mice were inoculated with other sheep hyperimmune or normal serum globulins. Anti-interferon globulin appeared to act in vivo because incubation of this globulin with peritoneal macrophages during the period of cell attachment or during the 18 hr after virus absorption did not render these cells permissive for VSV. Injection of mice with anti-interferon globulin did not affect the binding and uptake of labeled VSV by peritoneal macrophages. Although the underlying mechanism of this phenomenon is unknown, the results suggest that there may be low levels of endogenous interferon that contribute to host defense by maintaining some cells in an antiviral state.