Two in vitro systems were employed to delineate the estrogenic activity of daidzein (Da), alone or in combination with high or low concentrations of estrogen in two cell types possessing different estrogen-receptor (ER) isoforms, ERα and/or ERβ: (1) vitellogenin II (VTG), the egg yolk precursor protein and the endpoint biomarker for estrogenicity, in chicken primary hepatocytes, and (2) CHO-K1 cells transiently co-transfected with ERα or ERβ and estrogen-response elements (ERE) linked to a luciferase reporter gene. Da (100μM) alone induced VTG mRNA expression in chicken hepatocytes, albeit with much less potency compared to estradiol (E 2 ). Da exhibited different effects in the presence of 1μM and 10μM E 2 . At a concentration of 100μM, Da enhanced 1μM E 2 -induced VTG transcription by 2.4-fold, but significantly inhibited 10μM E 2 -induced VTG mRNA expression in a dose-dependent fashion from 1 to 100μM. Tamoxifen completely blocked the estrogenic effect of daidzein, alone or in combination with 1μM of E 2 , but did not influence its anti-estrogenic effect on 10μM E 2 -induced VTG mRNA expression. Furthermore, neither E 2 nor daidzein, alone or in combination, affected ERα mRNA expression, yet all the treatments significantly up-regulated ERβ mRNA expression in chicken hepatocytes. E 2 effectively triggered estrogen-response elements (ERE)-driven reporter gene transactivation in CHO-K1 cells expressing ERα or ERβ and showed much greater potency with ERα than with ERβ. In contrast, daidzein was 1000 times more powerful in stimulating ERβ- over ERα-mediated transactivation. Daidzein, in concentrations ranging from 5nM to 50μM, did not affect ERβ-mediated transactivation induced by 1nM E 2 , but it significantly inhibited ERβ-mediated transactivation induced by 10nM E 2 at 500nM. Despite the tremendous difference in sensitivity between the two in vitro systems, daidzein exhibited greater potency as an estrogen-antagonist for ERβ-mediated activity.