We previously reported that the bacterial immunomodulator CANTASTIM inhibited the LPS-induced TNF-alpha production in murine macrophages both in vivo and in vitro. In this report, we compared the activity of CANTASTIM with that of two phospholipids (cardiolipin and phosphatidylethanolamine) which are among the components of its lipid fraction. We noticed a significant reduction in the production of TNF-alpha upon stimulation with LPS in murine peritoneal macrophages pretreated for at least 3 h with CANTASTIM or cardiolipin. CANTASTIM was active at much lower concentrations than cardiolipin. Preliminary experiments with partially deacylated CANTASTIM indicated some decrease of TNF-alpha secretion. However, further studies are necessary to clarify this matter. Also, while CANTASTIM and its partially deacylated derivative could trigger the TNF-alpha secretion in murine macrophages, individual phospholipids did not. Based on these results, we concluded that CANTASTIM could induce the TNF-alpha suppression by multiple mechanisms, including the induction of regulatory cytokines such as IL-10 and CD14 receptor blockade/downregulation.