Phytoestrogens may function as partial agonists or antagonists of estrogen in many tissues including bone. Five phytoestrogens, belonging to the isoflavones and the flavonoids groups, were assayed in the human MG-63 osteoblastic cell line for their ability to stimulate transcriptional activity of an estrogen-response element (ERE)-luciferase reporter gene via the estrogen receptor β (ERβ). Although MG-63 cells were shown to express endogenous estrogen receptors, estradiol (E₂) did not affect transcriptional activity of an ERE reporter in these cells. However, E₂ did activate the ERE-reporter significantly in MG-63 cells where ERβ was overexpressed. The isoflavones, genistein and daidzein, caused a dose-dependent increase in the ERE-reporter activity in MG-63 cells overexpressing ERβ. Among the flavonoids, kaempferol activated ERE-reporter activity, whereas puerarin inhibited ERE-reporter transcription in cells overexpressing ERβ. Quercetin had no effect on ERE-reporter activity over a concentration range of 10⁻¹⁰–10⁻⁶ mol/l. The ERE-reporter activity induced by daidzein, genistein, and kaempferol was blocked by both ICI 182780 and 4-hydroxytamoxifen and partly blocked by puerarin. Our results demonstrated that different phytoestrogens exhibited differential transcription activity of an ERE-reporter via ERβ-mediated mechanisms in MG-63 cells.