A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.