The activation mechanism of estrogen receptor (ER) signaling by association with the aryl hydrocarbon receptor (AhR) was elucidated recently (Ohtake, et al., Nature 2003, 423, 545). In the present study, we established a reporter yeast strain to evaluate this ER signaling by association with the activated AhR. This yeast strain expresses human ER and AhR, and has a reporter plasmid with estrogen response elements. With this yeast strain we assayed ER activation by various AhR ligands, i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene, 3-methylcholanthrene, beta-naphthoflavone, and indirubin. All these ligands induced ER activation dose-dependently and their EC50 values were 60, 180, 130, 26, and 0.5 nM, respectively. Then, we measured the activity in water collected at 5 localities in the Ishizu River system in Japan. The activities of water samples ranged from 4.8 pmol/L (1.3 ng/L) to 52 pmol/L (14 ng/ L) (17beta-estradiol (E2) equivalent). These values were higher than those measured with the yeast for ER activation through direct ligand binding to ER. The direct ER ligand binding activities of the water samples ranged from 2.5 to 5.3 pmol/L (E2 equivalent). We also measured AhR activation of the water samples using a reporter yeast for AhR ligand activity. The activities ranged from 102 to 472 pmol/L (beta-naphthoflavone equivalent). These results indicate that the water samples contain substances that bind to AhR, and these substances contribute to ER signaling through AhR activation in the yeast reporter strain. This yeast reporter strain should be a useful tool to evaluate direct and indirect ER activation by environmental samples.