The estrogen receptor (ER)alpha is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERalpha 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. We found that truncating the last 16 residues of the F domain altered the activity of the human ERalpha (hERalpha) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins ("monobodies") that specifically bind to ERalpha/ligand complexes 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 wild-type 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 beta-strand-like structure. 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 ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERalpha ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERalpha selectively modulate its activity and its interactions with other proteins.