The bone sialoprotein (Bsp) gene provides an excellent model for studying mechanisms controlling osteoblast-specific gene expression. Although the RUNX2 transcription factor directly regulates many osteoblast-related genes, its function in Bsp expression remains uncertain. By using chromatin immunoprecipitation (ChIP) analysis in MC3T3-E1 (clone MC-4) preosteoblast cells, RUNX2 was shown to bind a chromatin fragment containing the proximal Bsp promoter. Two putative RUNX2-binding sites (R1 and R2) were identified within this region of the mouse, rat, and human genes and were shown to bind RUNX2 in vitro and in vivo (by ChIP assay). Site-specific mutagenesis established that both sites act as osteoblast-specific transcriptional enhancers and together account for nearly two-thirds of the total promoter activity. In addition, functional cooperativity was observed between the R2 site and an adjacent homeodomain protein-binding site previously characterized by this laboratory (the C site). All three sites (R1, R2, and C) are necessary for maximal promoter activity in osteoblasts. DLX5 in MC-4 cell nuclear extracts binds to the C site in vitro. Furthermore, ChIP assays revealed that DLX5 is selectively associated with chromatin in the vicinity of the C site only when Bsp is transcriptionally active. Finally, co-immunoprecipitation assays detected a physical complex containing DLX5 and RUNX2. Taken together, our data show that RUNX2 is a direct regulator of Bsp in osteoblasts and that it functions in cooperation with DLX5 or a related factor to activate osteoblast-specific gene expression.