ER serine 118 (Ser118) phosphorylation modulates AF1 function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cdk7 to catalyze this event. Here we show robust cdk7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E₂) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wt ER and either of two helix 12 mutants suggesting AF2-independent action, likely via shedding of HSP90. With E₂ treatment the predominantly nuclear, phosphorylated ER in COS-1 cells is detergent-soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118 phosphorylated ER is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. SERMs elicit a mixed response with phosphorylated ER in both detergent soluble and insoluble compartments. Apparent ligand-induced loss of ER protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118 phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ER degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the MMP1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ER can be AF2-independent. Further, they identify translocation of Ser118-phosphorylated ER out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.