The culture medium from several murine macrophage‐like cell lines contained a mitogenic activity that functioned synergistically with platelet‐poor plasma to induce DNA synthesis in quiescent density‐inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D1 (and other established lines of) macrophage‐like cells that were cultured either in medium alone or in medium supplemented with platelet‐poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage‐like cells were maintained in the medium. Serum‐free medium conditioned by macrophage‐cells did not stimulate DNA synthesis in density‐inhibited 3T3 cells in the absence of plasma; however, a transient (4‐hr) exposure to serum‐free macrophage‐conditioned medium allowed quiescent cells to respond to plasma‐derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage‐conditioned medium brought about DNA synthesis after a 12‐hr lag. The mitogenic activity that was in macrophage‐conditioned medium bound to DEAE‐Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage‐derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE‐Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin‐I (lymphocyte activating factor). Copyright © 1982 Wiley‐Liss, Inc.