Runx2 (previously known as Cbfal/Pebp2alphaA/AML3), a key transcription factor in osteoblast differentiation, has at least two different isoforms using alternative promoters, which suggests that the isoforms might be expressed differentially. Haploinsufficiency of the Runx2 gene is associated with cleidocranial dysplasia (CCD), the main phenotype of which is inadequate development of calvaria. In spite of the biological relevance, Runx2 gene expression patterns in developing calvaria has not been explored previously, and toward this aim we developed three probes: pRunx2, which comprises the common coding sequence of Runx2 and hybridizes with all isoforms; pPebp2alphaA, which specifically hybridizes with the isoform transcribed with the proximal promoter; and pOsf2, which hybridizes with the isoform transcribed with the distal promoter. These probes were hybridized with tissue sections of mouse calvaria taken at various time points in development. Runx2 expression was localized to the critical area of cranial suture closure, being found in parietal bones, osteogenic fronts, and sutural mesenchyme. Pebp2alphaA and Osf2 showed tissue-specific expression patterns. The sites of Pebp2alphaA expression were almost identical to that of pRunx2 hybridization but expression was most intense in the sutural mesenchyme, where undifferentiated mesenchymal cells reside. The Osf2 isoform was strongly expressed in the osteogenic fronts, as well as in developing parietal bones, where osteopontin (OP) and osteocalcin (OC) also were expressed. However, in contrast to Pebp2alphaA, Osf2 expression did not occur in sutural mesenchyme. Pebp2alphaA also was expressed prominently in primordial cartilage that is found under the sutural mesenchyme and is not destined to be mineralized. Thus, Osf2 isoforms contribute to events later in osteoblast differentiation whereas the Pebp2alphaA isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts.