We have recently demonstrated suppressor cells in spleens of mice bearing tumors induced by Moloney murine sarcoma virus (MSV) which were non-T cells and inhibited phytohemagglutinin Molney (PHA)-induced DNA synthesis of syngeneic normal spleen cells. From the present study, the suppressor cells appeared to be macrophages since they were radioresistant, inactivated by carrageenan, and removed by adherence columns and an iron/magnet technique. We have also found that suppressor cells were still fully active when added 16 hr after the mitogen, thus indicating that early mitogen-induced changes were not the target of suppressive action. It appeared that suppressor cells inhibited metabolic events related to the initiation of DNA synthesis and that they had a selective effect on proliferation-dependent lymphocyte effector functions. PHA-induced cytotoxic reactivity which in our system is largely independent of DNA synthesis was not depressed but actually enhanced in MSV spleens. Cytotoxicity of MSV spleen cells against syngeneic lymphoma cells was unaffected by suppressor cells whereas lymphocytes stimulation by mitomycin C-treated syngeneic lymphoma cellls was inhibited. MSV spleen cells also inhibited DNA synthesis of cultured murine lymphoma cells. This function was only slightly diminished after treatment with anti-omicron serum plus guinea pig complement. Furthermore, spleen cells from MSV tumor-bearing nude mice were as effective as spleen cells from their heterozygous littermates, thus suggesting that T lymphocytes are not the main effector cells of inhibition of lymphoma cell DNA synthesis. The inhibitor cells were radioresistnant, inactivated by carrageenan, and removed by adherence columns and the iron/magnet technique. These data strongly suggest that the inihibitor cells of lymphoma cell DNA synthesis are macrophages and that they belong to the same group of cells as the suppressor cells of PHA-induced lymphocyte proliferation.