This study investigated the potential species-specific estrogenic receptor (ER) activity of several natural and synthetic chemicals for bacterially expressed fusion proteins using a semi high throughput competitive binding assay. The fusion proteins consisting of the D, E and F domains of human (a ), mouse (a ), chicken, green anole and rainbow trout ER linked to the glutathione-S-transferase (GST) protein were prepared and partially purified. Saturation ligand-binding analysis of GST-hERa def (human), GST-mERa def (mouse), GST-cERdef (chicken), GST-aERdef (green anole) and GST-rtERdef (rainbow trout) fusion proteins revealed a single high affinity binding component for E2 with dissociation constants (K_d) ranging from of 0.3 to 0.9 nM.Although, the ERs from the different species exhibited similar binding preferences and binding affinities for many of the compounds examined, several differences in absolute and relative binding affinities were observed. Of the 35 chemicals examined, 4-hydroxytamoxifen was found to bind with greatest affinity for the ERs of all 5 species.The ranking of the potency of the xenobiotics and phytoestrogens varied among species with no predictable patterns and many examples of species-specific ligand preference.For example, a-zearalenol (IC₅₀ = 1.5 ± 0.7 x 10^-9 M) competed for binding with greater affinity than E2 for GST-rtERdef which was in contrast to all other GST-ERdef fusion proteins examined.However, the phytoestrogen coumestrol (IC₅₀ = 1.6 ± 0.3 x 10^-6 M) bound with lowest affinity to GST-rtERdef, while genistein (IC₅₀ = 3.0 ± 2.0 x 10^-8 M) bound with greatest affinity to GST-aERdef.These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic substances and that these differences may be due to the variability in the amino acid sequence within the ER ligand binding among species.