P388D1, a mouse macrophagelike cell line, was adapted to grow continuously in an unsupplemented, serum-free culture medium and continued to elaborate substances that were mitogenic for quiescent mouse fibroblasts (BALB/c 3T3 cells) and for thymocytes suboptimally stimulated with lectins. We have previously described  the fibroblast mitogenic activity as a macrophage-derived competence factor (MDCF). Serum-free, macrophage-conditioned culture medium was concentrated 1,000-fold by a combination of ultrafiltration (hollow fiber) and lyophilization. Concentrates of medium were subjected to gel filtration (Sephadex G-75 or G-150), and the fractions were assayed for mitogenic activity (MDCF) on density-arrested BALB/c 3T3 cells and for Interleukin-1 (IL-1) activity in suboptimally stimulated (Con A) mouse thymocytes. The apparent molecular weight (MW) of MDCF activity was estimated at 56,000 daltons, whereas the peak of IL-1 chromatographed at an apparent MW of 14-16K daltons. There was no detectable IL-1 activity in the MDCF fractions and no detectable MDCF in the IL-1 fractions. These data indicate that P388D1 cells produce both MDCF and IL-1 activities under continuous serum-free conditions and that the two activities are not identical. Stimulation of responsive mononuclear phagocytes with lipopolysaccharide and/or lymphokine-rich supernates resulted in a differential modulation of MDCF and IL-1 activities. Finally, antibody-purified IL-1 had no significant ability to stimulate DNA synthesis in quiescent fibroblasts at concentrations that were mitogenic for thymocytes. However, IL-1 did augment the mitogenic activity of suboptimal amounts of platelet-derived growth factor (PDGF), another competence factor. Further studies revealed that neither the generation nor the activity of MDCF was modulated by the presence of various inhibitors of proteolytic enzymes.