This study investigated the effects of E2, diethylstilbestrol (DES), antiestrogens, the phytoestrogen resveratrol, and the xenoestrogens octylphenol (OP), nonylphenol (NP), endosulfan, kepone, 2,3,4,5-tetrachlorobiphenyl-4-ol (HO-PCB-Cl₄), bisphenol-A (BPA), and 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on induction of luciferase activity in breast cancer cells transfected with a construct (pSp1₃) containing three tandem GC-rich Sp binding sites linked to luciferase and wild-type or variant ERα. The results showed that induction of luciferase activity was highly structure-dependent in both MCF-7 and MDA-MB-231 cells. Moreover, RNA interference assays using small inhibitory RNAs for Sp1, Sp3 and Sp4 also demonstrated structure-dependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. These results demonstrate for the first time that various structural classes of ER ligands differentially activate wild-type and variant ERα/Sp-dependent transactivation, selectively use different Sp proteins, and exhibit selective ER modulator (SERM)-like activity.