The secretory physiology of the T cell-produced lymphokine, mixed leukocyte rection suppressor factor (MLR-TsF), was characterized with respect to its kinetics of secretion and its sensitivity to a variety of metabolic blocking agents. It was found that spleen cells from alloantigen-immunized mice released active MLR-TsF after freeze-thaw lysis. Upon restimulation with the same priming alloantigen, MLR-TsF was secreted into culture supernatants, and the rate of secretion was determined to be nearly constant. Although colchicine and vinblastine, which bind to microtubules, are known inhibitors of lectin-induced proliferation, it was demonstrated that these drugs had no effect on the secretion of MLR-TsF. However, cytochalasin B, an inhibitor which also binds to some cytoskeletal and membrane-associated proteins, did inhibit the production of MLR-TsF. The above findings indicated that the activation-secretion mechanism of MLR-TsF was much like that described for lymphotoxin and macrophage migration inhibition factor. The dissociation between DNA synthesis and lymphokine secretion was also demonstrated in the MLR-TsF system. DNA synthesis plays no role in the in vitro production of suppressor factor, as determined by resistance to treatment with mitomycin C and γ-irradiation. However, new protein synthesis is required as indicated by the potent inhibitory effects of cycloheximide. Experiments utilizing timed addition and removal of cycloheximide defined a broad period of drug sensitivity, starting from the beginning of culture and lasting for 12 to 16 hr. In addition, experiments measuring the effect of cycloheximide on the MLR-TsF content of cell lysates demonstrated that the cell-associated lymphokine activity is lost when protein synthesis is interrupted. These experiments support the conclusion that MLR-TsF is synthesized de novo in culture. In addition, the secretory process itself may require protein synthesis.