Porphyromonas gingivalis lipopolysaccharide (LPS) and its bioactive center, lipid A, are known to exhibit very low endotoxic activities and activate LPS-hyporesponsive C3H/HeJ mice that have a point mutation in the cytoplasmic portion of Toll-like receptor (TLR) 4, in contrast to classical enterobacterial LPS and their lipid A. In the present study, we attempted to determine which TLR mediates the response to lipid A from P. gingivalis strain 381. P. gingivalis LPS and its natural lipid A fraction induced NF-kappa B activation primarily in Ba/F3 cells expressing mouse TLR 2 (Ba/mTLR2), rather than in those expressing mouse TLR4 and its accessory protein MD2 (Ba/mTLR4/mMD2). Further purification of the natural lipid A fraction resulted in a significant decrease of NF-kappa B activation in Ba/mTLR2, although not in Ba/mTLR4/mMD2. The synthetic counterpart of P. gingivalis strain 381-lipid A (compound PG-381) also elicited NF-kappa B activation in Ba/mTLR4/mMD2, but not Ba/mTLR2. Furthermore, P. gingivalis purified natural lipid A and compound PG-381 lacked the ability to activate gingival fibroblasts from C3H/HeJ, TLR4 knockout (KO) and myeloid differentiation factor 88 (MyD88) KO mice. These findings demonstrate that the P. gingivalis lipid A molecule induces cell activation via a TLR4/MD2-MyD88-dependent pathway, and suggest the possibility that unknown bacterial components in P. gingivalis LPS and its lipid A may induce cell activation via TLR2.