Fibroblast growth factor 2 (FGF-2) is an important regulator of bone formation and osteoblast activity. However, its mechanism of action on bone cells is largely unknown. A major route for FGF signaling is through the mitogen-activated protein kinase (MAPK) pathway. We showed recently that this pathway is important for activation and phosphorylation of Cbfa1/ Runx2, an osteoblast-related transcription factor (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). The present study examined the mechanism of FGF-2 regulation of the mouse osteocalcin gene in MC3T3-E1 preosteoblastic cells. FGF-2 stimulated osteocalcin mRNA and promoter activity in a dose- and time-dependent manner in MC3T3-E1 preosteoblastic cells. Similar results were obtained in mouse bone marrow stromal cells. This stimulation required Runx2 and its DNA binding site in the osteocalcin promoter. FGF-2 also dramatically increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) followed by phosphorylation of Runx2. Furthermore, a specific ERK1/2 phosphorylation inhibitor, U0126, completely blocked both FGF-2-stimulated Runx2 phosphorylation and osteocalcin promoter activity, indicating that this regulation requires the MAPK pathway. Deletion studies showed that the C-terminal PST domain of Runx2 is required for the FGF-2 response. This study is the first demonstration that Runx2 is phosphorylated and activated by FGF-2 via the MAPK pathway and suggests that FGF-2 plays an important role in regulation of Runx2 function and bone formation.