When macrophages derived from rat bone marrow were cultured in the presence of polyanions such as acetyl lignin (EP3), sulfonyl lignin (LS) or dextran sulfate (DS), the cells secreted TNF-α, IL-8 and nitric oxide (NO). EP3 had a dose-dependent effect on the secretion of TNF-α, IL-8 and NO. EP3 significantly affected secretion at concentrations greater than 5μg/ml. The EP3 effect was at its maximum between concentrations of 50 and 100μg/ml. LS and DS induced a slight increase in the secretion of cytokines and NO at a concentration of 100μg/ml. The use of the reverse-transcription polymerase chain reaction (RT-PCR) showed that the increases in cytokine and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA. Anti-TNF-α antibodies partially inhibited NO secretion by EP3-activated macrophages, although IL-8 secretion was independent of antibody treatment. The secretion of TNF-α and NO was also unaffected by the addition of anti-IL-8 antibodies. The addition of interferon-γ (IFN-γ) to the culture medium did not alter TNF-α and NO secretion by the EP3-activated macrophages, however, IL-8 secretion was increased when a low concentration of IFN-γ (0.2U/ml) was added, but was reduced in the presence of a high concentration of IFN-γ (2000U/ml). IFN-γ produced similar effects on cytokine and NO secretion in macrophages activated with lipopolysaccharide (LPS). Therefore, it is concluded that macrophages treated with polyanions secrete cytokines and NO, and that INF-γ is involved in the regulatory mechanism of cytokine and NO secretion.