4-Hydroxytamoxifen (4-OHT), a selective estrogen receptor modulator, is an agonist at a transforming growth factor-alpha (TGF-alpha) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ERalpha. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YERalpha enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YERalpha is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YERalpha complex. Unlike the estradiol-ERalpha complex, agonist activity of 4-OHT and raloxifene through ERalpha and D351YERalpha were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ERalpha, 4-OHT-D351YERalpha, and Ral-D351YERalpha. To address the role of AF1, stable transfectants of ERalpha or D351YERalpha with an AF1 deletion (D351deltaAF1, D351YdeltaAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351-3m, D351Y3m) or the COOH-terminal 537 deletion (D351delta537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ERalpha complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ERalpha complex.