A comparative study was undertaken to investigate the in vitro estrogenic activities (i.e. ligand binding, reporter gene induction) of a set of structurally diverse, synthetic and natural chemicals that included steroids, pharmaceuticals, phytoestrogens, environmental contaminants, pesticides and industrial chemicals. Relative binding affinity was determined at 4oC by assessing the ability of the test chemicals (1 nm to 10 µM) to compete with [3H]17_-estradiol (E2) for binding to bacterially expressed fusion proteins consisting of the D, E and F domains from the human (h)(_), the mouse (_), the chicken, the reptilian green anole (Anolis carolinensis, ac) and the rainbow trout (rt)(Onchorhynchus mykiss) estrogen receptors (ER) linked to glutathione S-transferase (GST). All GST-ERdef fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) ranging from 0.4 to 0.9 nM. Although, each fusion protein exhibited similar ligand preferences and relative binding affinities for many of the test chemicals, notable differences were observed with some compounds. In general, GST-rtER_def exhibited a preference for xenobiotics while GST-acER_def exhibited a preference for phytoestrogens, relative to the other ER fusion proteins. ER-mediated reporter gene induction was examined in MCF-7 cells transiently transfected with Gal4-ERdef constructs and a Gal4-regulated luciferase reporter gene. There was a lack of correlation between relative binding affinity and reporter gene induction for the test chemicals even though E2 induced reporter gene activity was comparable amongst the constructs (EC50 values - 0.05 to 0.7 nM). However, Gal4-rtERdef exhibited a significantly larger EC50 value of 28 nM at 37oC. Surprisingly, at 20oC, the Gal4-rtERdef EC50 value decreased to 1 nM. Saturation analysis confirmed that the Kd value was significantly lower at 4oC compared to 37oC indicating that GST-rtERdef ligand binding and reporter gene induction is temperature sensitive. A comparison of amino acid sequences identified two conservative residue differences in the ligand binding pocket of hERdef alpha (L349, M528) and rtERdef E domains. The effect of these substitutions on ligand binding and transactivation were examined by constructing reciprocal mutants, which effectively exchanged the binding pockets of the rtER and hER_.. The rtERdef M317L:I496M double mutant exhibited improvided E2 binding affinity and transactivation ability at higher temperatures, and displayed adopted the hERa phenotypic behavior for selected test chemicals that previously showed a species preference. These studies demonstrate that conservative changes in amino acid residue hydrophobicity and volume can significantly affect ER ligand binding and transactivation ability in a temperature dependent manner.