RANKL is a tumor necrosis factor (TNF)-like factor secreted by mesenchymal cells, osteoblast derivatives, and T cells that is essential for osteoclastogenesis. In osteoblasts, RANKL expression is regulated by two major calcemic hormones, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and parathyroid hormone (PTH), as well as by several inflammatory/osteoclastogenic cytokines; the molecular mechanisms for this regulation are unclear. To identify such mechanisms, we screened a DNA microarray which tiled across the entire mouse RankL gene locus at a 50-bp resolution using chromatin immunoprecipitation (ChIP)-derived DNA precipitated with antibodies to the vitamin D receptor (VDR) and the retinoid X receptor (RXR). Five sites of dimer interaction were observed on the RankL gene centered at 16, 22, 60, 69, and 76 kb upstream of the TSS. These regions contained binding sites for not only VDR and RXR, but also the glucocorticoid receptor (GR). The most distant of these regions, termed the distal control region (RL-DCR), conferred both VDR-dependent 1,25(OH)(2)D(3) and GR-dependent glucocorticoid (GC) responses. We mapped these activities to an unusual but functionally active vitamin D response element and to several potential GC response elements located over a more extensive region within the RL-DCR. An evolutionarily conserved region within the human RANKL gene contained a similar vitamin D response element and exhibited an equivalent behavior. Importantly, hormonal activation of the RankL gene was also associated with chromatin modification and RNA polymerase II recruitment. Our studies demonstrate that regulation of RankL gene expression by 1,25(OH)(2)D(3) is complex and mediated by at least five distal regions, one of which contains a specific element capable of mediating direct transcriptional activation.