We have studied how 2-arylindene systems, unsymmetrical nonsteroidal estrogens, orient themselves within the binding site of the estrogen receptor, relative to estradiol, by making a comprehensive comparison of the binding affinity of 16 analogues. These analogues are representatives of two major classes, those substituted at C-3 with an ethyl or with a phenyl substituent; within each class there are members that have different patterns of hydroxyl group substitution and C-1 oxo or alkyl substitution. Orientational preferences were inferred from the relative binding affinities and were supplemented by computer graphic molecular overlap studies that utilized crystal structures of selected representative compounds and the known tolerance of the estrogen receptor to substituents on the steroidal ligand estradiol. 2-Arylindenes with a 3-aryl substituent appear to orient with the indene system mimicking the A- and B-rings of estradiol (indene/AB mode). This orientation is supported by the fact that hydroxyl substitution at C-6 in the indene markedly elevates binding relative to hydroxyl substitution at the para position of the 2-phenyl substituent. A C-1 oxo substituent increases binding further, but a C-1 alkyl group has little effect. By contrast, the 2-arylindenes with a C-3 ethyl substituent appear to bind with the pendant C-2 ring, mimicking the A-ring of estradiol (pendant/A mode), as hydroxyl substitution in this ring elevates binding relative to the C-6 hydroxy analogues. C-1 alkyl substitution elevates binding affinity in this series; such a substituent in a C-1 S configuration would be projected into the receptor region normally occupied by the high-affinity 7 alpha- or 11 beta-alkyl estradiols. A C-1 oxo substituent produces only a modest binding enhancement in the C-3 ethyl series. A thermodynamic evaluation of receptor fit suggests that the smaller 3-ethyl-2-arylindenes are more efficient than the 2,3-diarylindenes in the use of the molecular bulk to achieve receptor binding. This analysis of the orientational preference of 2-arylindene nonsteroidal estrogens has important implications in the design of donor/acceptor-substituted 2-arylindenes as fluorescent ligands for the estrogen receptor.