Receptor activator of nuclear factor-kappaB ligand (RANKL) is a TNF-like factor that is both produced by osteoblasts, mesenchymal cells, and activated T cells and required for osteoclast maturation and survival. The gene is up-regulated by the two primary calcemic hormones, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and PTH. Previous studies have indicated that five enhancer regions located significantly upstream of the mouse Rankl transcriptional start site mediate up-regulation by 1,25(OH)2D3 and PTH. The most distal of these, termed mRLD5, is highly conserved in the human gene at -96 kb where it was also shown to be functionally active. Four additional mouse Rankl upstream enhancers are also highly conserved in the human gene at -20, -25, -75, and -87 kb. In the present studies, we characterized the activity of these regions, explored their capacity to mediate the actions of 1,25(OH)2D3, and identified the vitamin D response elements contained within the two most proximal segments. Interestingly, whereas the most distal of the five enhancers is the dominant mediator of 1,25(OH)2D3 activity in the mouse Rankl gene, that role in the human gene is manifested by the most proximal element at -20 kb. Importantly, activity at this region in response to 1,25(OH)2D3 was associated with a significant increase in histone acetylation as well as the enhanced recruitment of RNA polymerase II. Both likely reflect the primary role of this enhancer in human RANKL gene expression. Our studies confirm the complex nature of RANKL regulation and indicate that although the five enhancers are evolutionarily conserved across several species, their relative contributions to RANKL expression in response to 1,25(OH)2D3 may be different.