Periodontal disease is the multifactor disease. Lipopolysaccharide (LPS) has been shown as a major pathogenic substance. LPS derived from E. coli has been used to study for several decades according to its well-conserved structure typical for bacterial endotoxin, however, increasing information retrieve from using LPS derived from P. gingiva lis, which is recognized as a main pathogenic bacteria causing of periodontitis have shown different from that of E. coli. In addition to biological factor, physical factor may partially responsible for modulation of cell behavior. Oral cavity is often exposed to the change in temperature. Higher temperature above the body temperature may cause some effects on periodontal disease. The purpose of the present study was to investigate the effect of LPS; compared between that derived from P. gingiva lis and E. coli, and the physical environment; heat, on the expression of TNFU in human gingival and periodontal ligament fibroblasts in aspects of receptors and the intracellular signal transduction pathways. In the study, primary cultures of healthy gingival and periodontal ligament fibroblasts were treated with P. gingiva/is/ E. coli LPS or incubated at 45°C for thermal stimulation. Changes of TNFU at the levels of mRNA and protein were determined by using RT-PCR and ELISA. To elucidate the signal transduction pathway of P. gingivalis/ E. coli LPS and heatinduced TNFU, blocking anti-TLRs and TLRs siRNA were used to specify the required receptors. In addition, specific inhibitors of the intracellular signaling molecules were used to analyze the regulatory pathways. The result demonstrated that both PDL and GF cells could recognize both kinds of LPS by using either TLR2 or TLR4, but with the different extend, to induce TNFU expression. However, signaling pathway in P. gingiva lis LPS treated-PDL cells was passed through PI3K and Akt, whereas the pathway in E. coli LPS treated-PDL cells was via Akt and ERK. While pathway in P. gingiva/is LPS treated-GF cells was through ERK, p38 kinase and NF-kB, whereas the pathway in E. coli LPS treatedGF cells was via NF-kB only. Heat up-regulated TNFU was mediated by TRPVI. PKC was required for heat-induced TNFU expression. In addition, the use of Cytochalasin D, an inhibitor of actin polymerization, revealed that the cytoskeleton rearrangement might be an important mechanism for cellular sensing of thermal stimuli. In conclusion, all above the results suggest that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in PDL and GF cells, and both types of cells responded to LPS using the different pathway to up-regulate TNFU. Thermal stimulated-TRPVl activation led to the induction of TNFU mRNA expression in PDL cells suggesting role of heat in modulating of periodontal disease.