Although dura mater tissue is believed to have an important role in calvarial re-ossification in many in vivo studies, few studies have showed the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biologic activities exerted by dura mater on calvarial re-ossification (e.g. cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic co-culture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3 day-old) and adult (30 day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the co-culture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, while FGF-2 protein was not detectable in their conditioned media. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts.