Macrophage migration inhibitory factor (MIF) was found in media of human and mouse lymphocyte and fibroblast cell lines that were continuously growing. Its release was dependent on activation of the cells to enter the mitotic cycle, particularly on cells in S phase. The greatest quantity of MIF was detected in supernatants of lymphocytes collected during S phase after the cells were synchronized in G(1) and in supernatants of growing fibroblasts. When the latter were contact inhibited little or no MIF was found in media. MIF was also released into media of cells proliferating in homologous serum in the absence of fetal calf serum and into media lacking any protein. The MIF produced by lymphocyte lines eluted from Sephadex G-100 in the same fashion as MIF produced by the interaction of sensitized guinea pig cells and antigen. The results indicated that MIF is not a specific mediator of delayed hypersensitivity and cellular immunity and that MIF released by sensitized lymphocytes incubated with antigen merely reflects that fraction of cells activated by antigen to enter the mitotic cycle.