Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor- (TNF-), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-. The present study investigated the mechanisms involved in TNF- production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF- by E. faecalis. In addition, antioxidant treatment reduced TNF- production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF- expression by RAW 264.7 cells. Furthermore, activation of NF-xB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF- in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-xB, and AP-1.