A permanent cell line designated SL4c has been established from a primary culture of murine BALB/c spleen cells regularly stimulated with large doses of irradiated allogeneic cells plus exogenous interleukin-2(IL-2). After 8 months of cultivation, the cells of the SL4c line proliferate spontaneously and do not respond with an increase in proliferation to alloantigenic stimulation. The cells have the Lyt 1.2+, Lyt 2.2-, L3T4a+, Thy 1.2+ phenotype and exert a strong suppressive effect upon stimulation with freshly explanted cells. The SL4c line produces a suppressor factor (SF4c), which inhibits the mitogen-induced proliferation of normal lymphoid cells but does not suppress the proliferation of fibroblasts and sarcoma cells. The suppression is antigen non-specific, is not limited by H-2 restriction nor by interspecies barrier, and is not due to cytotoxic effect. However, the suppression is only detectable if the SF4c is added to the stimulated cells during the early stages of mitogen-induced proliferation. A tentative characterization of the relative molecular weight (MW) of the suppressor molecule based upon fractionation of SF4c supernatant on a Sepharose 6B column shows that the inhibitory activity is confined to the high MW fractions (300,000-350,000). Translation material obtained from Xenopus laevis oocytes, which were injected with RNA preparations isolated from SL4c cells, also shows the suppressive effect.