TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERalpha, ERbeta, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERalpha and ERbeta antagonist) or MPP (ERalpha antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERalpha and ERbeta was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERalpha and ERbeta was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10(-5) M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERalpha-dependent pathway by binding to this receptor and not by increasing the expression of ER.