The accumulated knowledge on the binding of estradiol (E2) and its analogs and the results of affinity-labeling studies have been reviewed and are used herein to derive a binding site model for the estrogen receptor (ER). Estradiol is nonpolar and hydrophobic, except at its molecular termini. Most of its skeletal flexibility resides in the B-ring, and it probably binds in a low-energy conformation. The phenolic OH group in the A-ring contributes about 1.9 kcal/mol to the binding free energy and probably acts primarily as a hydrogen bond donor. The 17 beta-hydroxyl group in the D-ring contributes approximately 0.6 kcal/mol to the binding and probably acts as a hydrogen bond acceptor, either directly or via a water molecule. There also seems to be a degree of flexibility in the region of the receptor that encompasses the D-ring. The aromatic ring contributes about 1.5 kcal/mol, probably through weak polar interactions with receptor residues that contact the beta-face of the steroid. The receptor seems to surround the ligand, so that all four rings contribute significantly to binding. Small hydrophobic substituents enhance binding affinity at positions 4, 12 beta, 14, and 16 alpha; whereas, larger hydrophobic substituents are tolerated at positions 7 alpha, 11 beta, and 17 alpha. In general, the ER is intolerant of polar substituents. Based on E2 analogs bearing affinity-labeling groups, cysteine residues might be present in the binding site in the area of C-4, C-17 alpha, and C-17 beta, and a lysine residue might be located near C-16. Models that represent the limits of deformability of the ligand binding site, the position of preformed pockets, and space occupied by the receptor are presented. The various elements in this model for the binding of steroidal estrogens by the estrogen receptor are consistent with evidence emerging from the crystal structures of related nuclear hormone receptor ligand complexes.