Some specific effects of E₂ are mediated by estrogen receptor beta (ERb); however, deciphering the actual role of the receptor is complicated by the discovery of five different subtypes (ERb1-5), arising from alternative splicing of ERb mRNA. Even though only ERb1 is activated by E₂, the other subtypes, dimerizing with ERb1, modulate its activity. ERb is mainly expressed in colon mucosa, prostate epithelium, bone, vascular endothelium and selected brain regions, where it controls specific physiological functions. It is now also clear that the presence of ERb in many tumors is associated to slow proliferation and better prognosis; characteristic of highly invasive neoplastic cells is variation of the relative expression of ERb subtypes with a decrease of ERb1, coupled to an increase of ERb2 and 5. Aim of the work was to clarify the specific role of ERb subtypes in health and disease, by using two synthetic and the natural ERb selective ligands in different cell lines transiently transfected with the various ERb subtypes and gene reporters. The results demonstrate that the relative potency of the natural vs synthetic ligands depends on the cell context, on the molecular parameter considered and on the relative expression of ERb subtypes. In conclusion, our data clearly show that ERb selective ligands constitute a useful tool to clarify the complex mechanism of action and the actual functions of this receptor in the various normal or transformed cells in which it is expressed.