When rat bone marrow macrophages were incubated with acetyl lignin (EP3) in the presence of a 10% solution of fetal bovine serum, the macrophages secreted tumor necrosis factor (TNF-α) in a dose-dependent manner. This was followed by macrophage multinucleation. EP3 was found to have a significant effect on TNF-α secretion at a minimum dose of 5 μ/ml and produced no significant further increase at levels above 50 μg/ml, while multinucleation was most active at 10 μg/ml. However, multinucleation did not occur at higher concentrations of EP3 (50 μg/ml and 100 μg/ml). Secretion of TNF-α was significantly reduced in the absence of fetal bovine serum, whereas multinucleation was very active, starting after 6 h of incubation. At concentrations of 100 μg/ml, sulfonyl lignin (LS) and dextran sulfate (DS) only induced low levels of TNF-α secretion from macrophages, but induced active multinucleation. The multinucleation induced by addition of LS or DS was inhibited by further addition of EP3. Thus, macrophage multinucleation was most active when a low level of TNF-α was secreted from the macrophages.