Although important roles of receptor activator of NF-kappaB ligand (RANKL) and its receptor (RANK) have been established for osteoclastogenesis and bone resorption, their expression and roles during physiological root resorption remain uncertain. Physiological root resorption for shedding of human deciduous teeth is mediated by osteoclast-like cells (odontoclasts). In this study, we examined the expression of RANKL and osteoprotegerin (OPG), a decoy receptor that prevents RANKL from binding to RANK in human periodontal ligament (PDL) cells during physiological root resorption using immunocytochemistry and reverse transcriptase polymerase chain reaction. The effect of RANKL on root resorbing activity of odontoclasts was evaluated by measuring the size of dissolved area on calcium phosphate-coated coverslips. The PDL cells isolated from either non-resorbing deciduous teeth or permanent teeth abundantly expressed OPG, but not RANKL. In contrast, PDL cells derived from resorbing deciduous teeth dominantly expressed RANKL. Human odontoclasts derived from resorbing deciduous teeth expressed both RANKL and RANK. It was observed that RANKL increased odontoclast actin ring formation and resorbing activity in a dose-dependent manner. These results indicate that PDL cells during the root-resorbing state express RANKL but decrease OPG expression. Expression of RANKL likely participates in odontoclastogenesis and activates physiological root resorption.