Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently-tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ER or ER in an in vitro high throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ER and ER with affinity ranging from 20 nM to 500 nM. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, while antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC₅₀) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen resulting in distinct EC₅₀ values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ER was observed in the presence of the ER-selective agonist DPN, but not the ER-selective ligand PPT. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.