Runx2 is a key transcription factor in osteoblast differentiation, and its activity is regulated by fibroblast growth factors (FGFs). Craniosynostosis, characterized by premature suture closure, results from mutations that generate constitutively active FGF receptors (FGFRs). We previously showed that FGF/FGFR-activated protein kinase C (PKC) is involved in the expression and activity of Runx2. Activated PKCdelta physically interacts with Runx2 in FGF2-stimulated MC3T3-E1 preosteoblastic cells. Immunopurified Runx2 protein reacted with PKCdelta kinase, and a phosphorylated 1460-Da peptide fragment (amino acids 241-252, 1380-Da) derived from Runx2 was also detected in MS analysis. Computer analysis predicted that Ser247 in this Runx2 can be a possible phosphorylation site by PKCdelta. We also showed that Runx2 activity after FGF stimulation correlates with the presence of the Runx2 Ser247 residue. The S247A (Ser --> Ala) mutation confers decreased transcriptional activity on a Runx2-responsive promoter after FGF treatment.