Cell line R453, established from a Rauscher virus-induced myeloid leukemia in a C57BL/6 mouse, was induced to differentiate in vitro into macrophages and granulocytes with ascitic fluids from animals bearing various ascites tumors or from mice treated with complete Freund's adjuvant, conditioned media from various cell lines, and glucocorticoid hormone. Differentiated R453 cells had a morphology similar to that of macrophages and granulocytes in normal hematopoietic organs, and they phagocytized small paricles such as latex particles, moved in soft agar showing locomotive activity, and had Fc and C3 receptors on the cell surface. This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of RNA synthesis (actinomycin D, nogalamycin, or chromomycin A3), protein synthesis (puromycin or cycloheximide), or DNA synthesis (methotrexate, hydroxyurea, 5-fluorodeoxyuridine, or 1-beta-D-arabinofuranosylcytosine) in the presence of ascitic fluid. Of the inhibitors, actinomycin D was the most effective at a low concentration (5 ng/ml) in stimulating induction of differentiation of R453 cells. However, these inhibitors alone did not induce differentiation of R453 cells. The factor(s) in ascitic fluid that stimulates differentiation of R453 cells was heat labile, nondialyzable, and inactivated by trypsin.