Receptor activator of NF-kappaB ligand (RANKL) is a membrane-bound signal transducer necessary for the induction and maintenance of osteoclasts. To clarify the molecular mechanism by which 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) augments osteoclasts, we characterized the promoter region of the mouse RANKL gene. Mirroring in vitro osteoclastogenesis demonstrated by a coculture of bone marrow macrophages with ST2 stromal cells, Northern blot, and nuclear run-on analyses showed that 1,25-(OH)(2)D(3) upregulate RANKL gene expression at the transcriptional level. Using a series of deletion mutants of mouse RANKL promoter-luciferase reporter gene constructs, transient transfection studies revealed that the inductive effect of 1,25-(OH)(2)D(3) was abolished when the region up to -723 was deleted. An electrophoretic motility shift assay demonstrated that the VDR-RXRbeta heterodimer bound to AGGTCAGCCTGGTTCA (-937/-922), and VDRE/nuclear protein supershift complexes that bound to anti-VDR and -RXRbeta antibodies were detected in the nuclear extract of 1,25-(OH)(2)D(3)-treated ST2 cells. Furthermore, induction of mutation to the putative VDRE also diminished the inductive effect of 1,25-(OH)(2)D(3). We therefore concluded that mouse RANKL gene is one of the target genes of 1,25-(OH)(2)D(3) containing a functional VDRE in the promoter region.