Our recent work showed that human periodontal ligament fibroblasts (HPLF) secrete bioactive osteoprotegerin (OPG), which inhibits osteoclastic differentiation and activity. However, it is unknown how HPLF regulate bone metabolism in the presence of lipopolysaccharide (LPS), which is a cell component of gram-negative bacteria and a pathogen in inflammatory bone diseases such as periodontitis. The present study examined the effects of Escherichia coli LPS on the gene expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), OPG, and receptor activator of NF-kappa B ligand (RANKL) in HPLF using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. In HPLF cultured with LPS for 48 h, expression of both OPG and RANKL mRNA was up-regulated, whereas for up to 24 h of stimulation, such up-regulation was not observed. However, LPS increased expression of IL-1 beta and TNF-alpha mRNA within 6 h of treatment. Moreover, in HPLF cultured with IL-1 beta or TNF-alpha, OPG and RANKL expression was induced within 12 h of culture. The administration of neutralizing antibodies against human IL-1 beta or TNF-alpha to LPS-treated cultures of HPLF inhibited the induction of OPG and RANKL expression. These suggest that LPS stimulates both OPG and RANKL expression in HPLF by up-regulating IL-1 beta and TNF-alpha. In addition, administration of conditioned medium (CM) from HPLF (HPLF-CM) stimulated with LPS for 48 h to mouse bone marrow culture failed to induce osteoclast-like cell (OCL) formation. When mouse spleen cells were cocultured with HPLF in the presence of LPS, OCL formation was completely blocked. Taken together, our results indicate that human periodontal ligament cells stimulated with LPS inhibit osteoclastogenesis by producing more effective OPG than RANKL via the induction of IL-1 beta and TNF-alpha.