Estrogens, acting through the estrogen receptors (ERs), play crucial roles in regulating the function of reproductive and other systems under physiological and pathological conditions. ER activity in regulating target genes is modulated by the binding of both steroidal and synthetic non-steroidal ligands, with ligand binding inducing ERs to adopt various conformations that control their interactions with transcriptional coregulators. Previously, we developed an intramolecular folding sensor with a mutant form of ER (ER_G521T) that proved to be essentially unresponsive to the endogenous ligand 17-estradiol, yet responded very well to certain synthetic ligands. In this study, we have characterized this G521T-ER mutation in terms of the potency and efficacy of receptor response towards several steroidal and non-steroidal ligands in two different ways: directly, by ligand effects on mutant ER conformation (by the split-luciferase complementation system), and indirectly, by ligand effects on mutant-ER transactivation. Full-length G521T-ER shows no affinity for estradiol and does not activate an estrogen-responsive reporter gene. The synthetic pyrazole agonist ligand PPT is 100-fold more potent than estradiol in inducing intramolecular folding and reporter gene transactivation with the mutant-ER, whereas both ligands have high potency on wild-type ER. This estradiol-unresponsive mutant-ER can also specifically highlight the agonistic property of the SERM, 4-hydroxytamoxifen, by reporter gene transactivation, even in the presence of estradiol, and it can exert a dominant negative effect on estrogen-stimulated wild-type ER. This system provides a model for ER-mutants that show differential ligand responsiveness to gene activation to gain insight into the phenomenon of hormone resistance observed in endocrine therapies of ER-positive breast cancers.