Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E2). Phytoestrogen concentrations used were 1000 times higher than for E2, except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E2 (DC50) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC50: 7 nM) > formononetin (DC50: 260 nM) > genistein (DC50: 570 nM) > equol (DC50: 5.3 μM) > daidzein (DC50: ) > biochanin A (DC50: 100 μM). In sturgeon, estradiol (DC50: 5 nM) > genistein (DC50: 220) > formononetin (DC50: 1 μM) > equol > (DC50: 8.3 μM) > daidzein > (DC50: 80 μM) > biochanin A (DC50: 100 μM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.