Signaling of nuclear receptors depends on the structure of their ligands, different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on ER and ER mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ER, ligands can be classified into three distinct groups: 1. Ligands that do not affect the mobility of the receptor; 2. ligands that cause a moderate effect and 3. ligands that strongly impact mobility of ER. Interestingly, we found that for ER such a classification was not possible as ER ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response towards the anti-estrogens ICI and raloxifene which was not attributable to differential sub-nuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ER, while ER retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteosome machinery. This novel finding that ER retains its mobility in the presence of anti-estrogens, could be important for its ability to regulate genes that do not contain classic ERE sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.