<jats:title>Abstract</jats:title><jats:p>The runt homology transcription factor Runx2/Cbfa1 is essential for bone development and osteoblast differentiation. Regulatory mechanisms that govern Runx2 transcription in osteoblasts define the osteogenic pathways that control skeletal development. In this study, we systematically examined transcription factor binding within the upstream Runx2 P1 promoter, which regulates expression of the bone‐related Runx2 factor. We identified two novel protein/DNA interactions that are mediated by sequence specific factors, based on cross‐competition experiments, point mutations, and gel‐shift immunoassays. One complex recognizes a non‐canonical Runx2 site, whereas the other factor binds to a palindromic sequence. Site‐directed mutagenesis of the novel Runx2 motif (5′TCCCAC3′) within the 0.6 kb rat Runx2 promoter reduces transcription by 2‐fold, indicating that this site supports enhancement of Runx2 promoter activity. Mutation of the palindromic motif (5′AGTACT3′) results in a 2–3‐fold activation of the Runx2 promoter, demonstrating that the wild type sequence contributes to transcriptional repression. These studies, together with our previous findings of auto‐suppression of the Runx2 promoter and negative regulation by 1,25(OH)<jats:sub>2</jats:sub> Vitamin D3, suggest that physiological control of Runx2 gene expression is mediated by a series of intricate regulatory mechanisms. J. Cell. Biochem. 86: 403–412, 2002. © 2002 Wiley‐Liss, Inc.</jats:p>