NOD1 (Card4) and NOD2 (Card15) are thought to be responsible for cytoplasmic defense against bacterial entry. To gain further knowledge about how their expressions are regulated in murine macrophages. we investigated the expression of NOD1 and NOD2 mRNAs after stimulation with various endotoxins, lipopolysaccharide, lipoteichoic acid and peptidoglycan. In macrophage RAW264.7 cells, the first and second rises in NOD1 and NOD2 mRNAs were observed at 2 hr and at 8-12 hr after endotoxin treatment. Increases in NOD1 and NOD2 mRNAs at 2 hr in lipopolysaccharide-treated RAW264.7 cells were reduced with the use of NF-kappa B inhibitor. caffeic acid phenetyl ester. In RAW264.7 cells, lipopolysaccharide-induced increases in NOD1 and NOD2 mRNAs were inhibited with anti-TLR4 antibody, and partially reduced in peritoneal macrophages obtained from TLR4-deficient mice. Furthermore. NOD I and NOD2 mRNA expressions in RAW264.7 cells were increased by the treatment with proinflammatory cytokines. tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or IL-6. In TNF-alpha deficient macrophages, the expression of NOD molecules was minimal at 12 hr, and the second rise in NOD mRNA seen in lipopolysaccharide-treated RAW264.7 cells was inhibited with anti-TNF-alpha. but not with anti-IL-1 beta or anti-IL-6 antibody. These observations suggest that immediate response of NODS to endotoxins could result from NF-kappa B activation via TLR signaling, whereas the second rise in NOD mRNAs might have resulted from TNF-alpha production possibly through NF-kappa B. TLR. and/or NOD signalings.