One-way mixed lymphocyte cultures employing human adenoid or peripheral blood lymphocytes activate lymphotoxin (LT)-secreting cells. Kinetic analysis of lymphocytes stimulated in mixed culture demonstrates that LT is secreted before the onset of DNA synthesis, but that maximum levels of LT secretion are reached simultaneously with maximum levels of DNA synthesis. Although the response of peripheral leukocytes is qualitatively similar to the response of adenoid-derived lymphocytes, unexplained high nonspecific background levels of toxic material(s) obscure early events in the former response. While cytochalasin B reversibly inhibits LT secretion, mitomycin C treated cultures are still capable of LT secretion. The results suggest that a population of cells exists, which does not require DNA synthesis to develop into effector cells. The requirement for DNA synthesis for the maximal development of effector cells may reside in a separate helper cell population as postulated by the two cell model of the mixed lymphocyte reaction. © 1975 Academic Press, Inc. All rights reserved.