RANKL, a protein essential for osteoclast development and survival, is stimulated by parathyroid hormone (PTH) via a PTH receptor 1/cyclic AMP (cAMP)/protein kinase A (PKA)/CREB cascade, exclusively in osteoblastic cells. We report that a bacterial artificial chromosome-based transcriptional reporter construct containing 120 kb of RANKL 5'-flanking region was stimulated by dibutyryl-cAMP in stromal/osteoblastic cells, but not other cell types. Full cAMP responsiveness was dependent upon a conserved 715-bp region located 76 kb upstream from the transcription start site, which we identified by sequential deletion analysis and by comparison of human and mouse genomic sequences in silico. This region contained conserved consensus sequences which bound CREB and the osteoblast-specific transcription factor Runx2, and when mutated blunted cAMP responsiveness. Overexpression of Runx2 potentiated cAMP responsiveness of the endogenous RANKL gene in a cell-type-specific manner. Lastly, PTH responsiveness of the endogenous RANKL gene was abrogated in mice from which we deleted this conserved upstream region. Thus, PTH responsiveness of the RANKL gene is determined by a distant regulatory region that responds to cAMP in a cell-type-specific manner and Runx2 may contribute to such cell-type specificity.