Nuclear receptors (NR) are a superfamily of ligand activated transcription factors that modulate specific gene expression. They are comprised of modular domains: The amino-termini of the receptors that have transcription transactivation functions; the centrally placed and highly conserved DNA-binding domain (DBD) which directs receptor binding to DNA; and the carboxy-terminal ligand-binding domain (LBD) that binds the ligand and mediate receptor-induced changes in transcriptional control. Estrogen receptor (ER) is classified as a Class I nuclear receptor. ER exerts diverse array of biological effects through interactions with estrogen and estrogen like molecules. For many years, it was believed that estrogen exerted it effects through a single receptor, now referred to as ER-a. More recently, a second estrogen receptor, ER-b, was identified. In this work we have two objectives: The first one is to clone ERa and ERb LBD into pET28a(+) and pSMT3 expression vector to obtain recombinant proteins to be used in structural studies; and the second one is to establish a transactivation assay “in vivo” for ERb which will allow us to look for new agonists or antagonists. We cloned ERaLBD and ERbLBD into pGEM-T vector after amplification of both domains by Polymerase Chain Reaction. The recombinant pGEM-T vectors were transformed into E.coli DH5a and positives colonies were purified, sequenced, and sub-cloned into pET28a(+) or pSMT3 expression vectors. Although protein expression was obtained in E. coli strain BL21 (DE3) at 37 ^oC for all constructs tested, only the fusion proteins, SUMO-ERaLBD and SUMO-ERbLBD, appeared to be soluble. Concomitantly, we have established the electroporation conditions for the transactivation luciferase based assay (0.34 kV and 750 mF) and the dose dependent curve for ERb-estradiol. The trasactivation assays are employed to test new synthetic ligands in search for possible new agonistic and/or antagonistic activities.