Allosteric communication between interacting molecules is fundamental to signal transduction and many other cellular processes. To better understand the relationship between nuclear receptor (NR) ligand positioning and the formation of the coactivator binding pocket, we investigated the determinants of ligand selectivity between the two estrogen receptor subtypes ERalpha and ERbeta. Chimeric receptors and structurally guided amino acid substitutions were used to demonstrate that distinct "hot spot" amino acids are required for ligand selectivity. Residues within the ligand binding pocket as well as distal secondary structural interactions contribute to subtype-specific positioning of the ligand and transcriptional output. Examination of other NRs suggests a mechanism of communication between the ligand and coactivator binding pockets, accounting for partial agonist and dimer-specific activity. These results demonstrate the importance of long-range interactions in the transmission of information through the ligand binding domain as well as in determining the ligand selectivity of closely related NR receptor subtypes.