Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T [thymus-derived] cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 h at 37.degree. C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. Memory T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, these cells persisted for at least 6 mo. Memory cells, like killer T cells, bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. When cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 h, but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 h of culture. Subsequent increases in the activity of 45-day cells were dependent upon cell proliferation. The antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte c[cyclic]AMP levels.