Runx2 is a master transcription factor in osteoblasts, yet its mechanism is poorly understood. In particular, there is a paucity of information about its target genes and their regulation. To address this, we first used ChIP Display to discover novel genomic targets occupied by Runx2 in living MC3T3-E1 osteoblastic cells. One of these targets was located within the promoter of Tram2, whose product facilitates proper folding of type I collagen. We demonstrated that Tram2 mRNA levels were altered by exogenous Runx2, and that this occurred in a BMP- and cell typedependent manner. Thus, Tram2 is likely a Runx2 target gene and may participate in its osteogenic function. Next, we measured endogenous Runx2 expression in MC3T3-E1 cells during development of the osteoblast phenotype, to see if it could explain the dramatic increase in mRNA levels of Osteocalcin (OC), a classic Runx2 target gene. Surprisingly, we discovered that it could not, as Runx2 expression decreased over time, along with in vitro DNA binding activity. Instead, developmental stimulation of OC by Runx2 is attributable to enhanced promoter occupancy in vivo. A remarkably similar pattern of recruitment was observed at the Glt28d2 promoter, a novel Runx2 genomic target discovered by ChIP-Chip analysis of cells in which the OC promoter is maximally occupied. Thus, Runx2 acquires the ability to access target genes relatively late during development of the osteoblast phenotype, and this is most likely due to activation of collaborating factors or to post-translational modification of Runx2 itself. Expanding our knowledge of Runx2 target genes and their regulation is warranted to better understand the regulation of osteoblast function and to provide opportunities for the development of new bone anabolics.