Supernatants from incubated normal mouse spleen cells suppressed the DNA synthetic response induced by polyclonal B‐ and T‐cell activators and the primary immune response to sheep erythrocytes in normal spleen cells in vitro. The inhibitory effects of the supernatants were found to be dependent on the culture system used. The main cell population producing or releasing the inhibitory factors was nonadherent spleen cells, but macrophages and bone marrow cells could also give supernatants with inhibitory effects. Thymocytes did nut release any inhibitors under the same conditions. Separation of the supernatants showed that there were it least two factors with inhibitory effects on proliferation in lymphocytes. One of these factors had a molecular weight of less than 10,000 (not degradable by protease) and suppressed activation by all tested polyclonal B‐ and T‐cell activators. Another factor (a protein with a molecular weight of more than 30, 000) inhibited activation by some polyclonal B‐cell activators (lipopolysaccharide, type III pneumococcal polysaccharide) whereas the proliferation induced by dextran sulfate Was less or not at all affected under the same culture conditions. Trivial mechanisms for inhibition by supernatant factors, such as medium depletion, general toxicity, and accumulation of free thymidine in the medium, were