In order to develop high affinity, fluorescent ligands for the estrogen receptor based on 2-arylindenes, it is important to understand how this non-steroidal estrogen is oriented within the binding site and to know how hydroxyl substituents affect binding. To investigate these issues a series of dihydroxyl-substituted 2,3-diphenylindenes were prepared by the cyclization of appropriately substituted alpha-benzyldesoxybenzoins, and their binding affinities for the estrogen receptor measured by a competitive radiometric binding assay. Introduction of a p-hydroxyl group in the 2-phenyl ring of two 2,3-diphenyl-6-hydroxyindene systems causes a 3-fold increase in binding affinity, whereas, p-hydroxylation in the 3-phenyl ring of these systems causes a 2-fold reduction in binding affinity. The parallel change in binding affinity in these two systems suggests a consistent binding orientation of the 2,3-diarylindene systems, which, on the basis of earlier studies, has the indene system corresponding to the A/B-ring system of estradiol. This orientation model and the enhanced affinity of the p-hydroxy 2-ring derivatives are suggestive of a new hydrogen bonding site below the D-ring binding site. Changes in receptor binding affinity upon hydroxylation in triphenylacrylonitrile ligands for the estrogen receptor, reported by others, do not show such parallelism, suggesting that different derivatives may not be bound in congruent orientations. A m-hydroxyl substituent in ring-3 of the 2,3-diarylindene has very little effect on receptor binding. In designing fluorescent 2,3-diarylindene ligands for the estrogen receptor, 3-ring hydroxylation may be useful in reducing non-specific binding and in modifying electron donation to the fluorophore with only modest or no reduction in binding affinity. p-Hydroxylation of the 2-ring, although increasing receptor binding, is not consistent with the electron accepting nature required of this ring.