Summary: Spleen cells from C57BL mice immunised once with L5178Y lymphoma cells, which are of DBA/2 genotype, were assayed for cytotoxicity by measuring their effect on the growth of L5178Y cells in vitro. Two populations of cytotoxic spleen cells were distinguished: (1) those found predominantly 6-12 days following immunisation; and (2) those found 14 days and later following immunisation. The cytotoxic activity of the “early” cells was relatively radioresistant and was not affected by drugs which inhibit nucleic acid synthesis. These early cells resemble those found in lymph from draining nodes, which are cytotoxic only 4-8 days after immunisation. Spleen cells taken from mice and rats 21 days after immunisation lost cytotoxic activity by exposure to 500 R of X-rays and to Mitomycin C, actinomycin D, and potassium cyanide. Removal of macrophages did not reduce the cytotoxicity of either class of spleen cell. Cytotoxicity was unaffected by the addition of a source of complement, but this does not exclude the participation of some complement components (which are synthesised by spleen cells). By following the release of 61Cr, it was shown that the L5178Y cells lysed sooner after the addition of spleen cells taken at 7 days, as compared to 21 days, after immunisation. The 21-day spleen cells acquired some of the properties of 7-day spleen cells (such as radioresistance) after exposure to target cells for 10 hr. The hypothesis is advanced that the cells responsible for cytotoxicity in spleens at 7 days after immunisation are immunoblasts (i.e., large pyroninophilic cells), as had earlier been shown to be the case for cells in the lymph from immunised donors. These cells, it is postulated, are directly cytotoxic, possibly because they already possess the machinery for the synthesis of antibody. The cells found in spleen 21 days after immunisation are only potentially cytotoxic and become transformed—hence their radiosensitivity—by contact with target cell antigen into the actual cytotoxic cells, which are presumably immunoblasts