Previously, we discovered that periodontal ligament (PDL) cells not only support osteoclastogenesis through cell-to-cell contact, but also inhibit the formation of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells by a producing soluble factor(s). Furthermore, PDL cells express both receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) messenger RNA (mRNA). Clinically, "ankylosed teeth," which lack periodontal ligament, cannot be moved with orthodontic tooth treatment. From this, we hypothesized that PDL cells under mechanical stress should play a pivotal role in osteoclast formation during orthodontic tooth movement. This study examined how mechanical stress affects the osteoclastogenesis-supporting activity of PDL cells. PDL cells were compressed continuously and then cocultured with peripheral blood mononuclear cells (PBMCs) for 4 weeks. PDL cells under mechanical stress up-regulated osteoclastogenesis from PBMCs. Furthermore, the expression of RANKL mRNA and protein in PDL cells increased with compressive force in parallel with the change in the number of osteoclasts. In addition, cyclo-oxygenase 2 (COX-2) mRNA expression was induced by compressive force, and indomethacin inhibited the RANKL up-regulation resulting from compressive force. PDL cells under compressive force exhibited significantly increased prostaglandin E2 (PGE2) production in comparison with control PDL cells. Exogenous PGE2 treatment increased RANKL mRNA expression in PDL cells. Interestingly, OPG expression remained constant throughout compressive force or PGE2 treatment. In conclusion, compressive force up-regulated RANKL expression in PDL cells. Furthermore, RANKL up-regulation in mechanically stressed PDL cells was dependent on PGE2.