The estrogen receptor alpha (ER) is a biologically and clinically important ligand-modulated transcription factor. We and others have shown that the F domain of the ER modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain on the responses of the hER to estradiol (E₂) and 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator SRC-1, the interactions of the ER ligand-binding domain (LBD) and F domain with a receptor-interacting domain (RID) of the coactivator SRC-1 or engineered proteins ("monobodies") that specifically bind to ER/ligand complexes, and expression of the endogenous pS2 gene. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the LBD and F domains specifically reduced the 4-OHT-dependent interactions of the hER LBD and F domains with monobodies. By contrast, truncating the last 16 residues of the F-domain altered the activity of the hER on an ERE-driven promoter in response to E2 or 4-OHT, its sensitivity to SRC-1 overexpression in mammalian cells, and its interaction with the SRC-1 RID and monobodies in the absence of ligand in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs the wt ER. The region includes a distinctive segment (residues 579-584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a -strand-like structure. These results show that regions within the F domain of the hER selectively modulate its activity and its interactions with other proteins.