Two tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated. In the one-way "mixed lymphocyte interaction" system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP), when low levels (0.01 to 0.001 mug per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 mug per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [125I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7 days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, free specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant "killer" cell activity with spleen cells from primed mice. In a syngeneic tumor system, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice.