Peritoneal exudate cells and splenic cells of mice infected with Listeria monocytogenes show increased production of interferon-gamma (IFN-gamma) after antigen or mitogen stimulation. When an IFN-gamma-specific enzyme-linked immunosorbent assay was used, increased production was first observed 2 days after infection in peritoneal cells and 4 to 6 days after infection in splenic cells. The increased production of IFN-gamma correlated with the clearance of Listeria from the peritoneal cavity and spleen. Macrophages derived from mice at these times were activated as evidenced by expression of nonspecific tumoricidal activity against 111In-labeled P815 mastocytoma cells. Injection of neutralizing monoclonal anti-IFN-gamma into 1-day-infected mice completely inhibited the generation of activated macrophages. Normal hamster IgG had no effect. In vivo, the monoclonal antibody also abrogated clearance of bacteria from the spleen and peritoneal cavity. Six days after injection of a sublethal dose of Listeria, the peritoneal cavity of control mice treated with normal hamster IgG was devoid of bacteria and the spleen contained less than 10(3) colony-forming units. However, mice treated with anti-IFN-gamma carried more than 8 X 10(6) colony-forming units in either anatomical site at day 6 and exhibited a higher mortality rate. These results indicate that IFN-gamma production is required for the in vivo generation of activated macrophages and the clearance of bacteria during Listeria infection.