The R4-6A2 rat-mouse hybridoma, which secretes a rat IgG1 monoclonal antibody (MAb) capable of neutralizing murine interferon-gamma (MuIFN-gamma), was grown as ascites in athymic nude (nu/nu) mice. The neutralizing titers of ascitic fluids were 400 times higher than those of supernatants from R4-6A2 cultures. Moreover, the specific activities (MAb) neutralizing units/mg protein) of the ascitic fluids were much greater than those of R4-6A2 culture supernatants. Ascitic fluid MAb was purified by anion-exchange chromatography, with excellent recovery of applied neutralizing activity. The ability of the R4-6A2 anti-MuIFN-gamma MAb to neutralize several different activities of MuIFN-gamma was studied. The MAb neutralized the ability of MuIFN-gamma to inhibit the growth of cells (antiproliferative activity). The ability of MuIFN-gamma to render cells of a heterologous species (rat) resistant to viral replication was also neutralized by this MAb. The MAb-neutralizing titers for MuIFN-gamma antiviral activities, on both homologous (murine L929) and heterologous (rat fibroblast) cells, were inversely proportional to the antiviral activity titers on each cell type.