Estrogenic biological effects are primarily mediated through the two estrogen receptor isoforms (ERα and ERβ). Three main mechanisms of action for ERα include the nuclear genomic actions, including the classical and “tethered”- mediated responses, and the non-nuclear, non-genomic, rapid action responses. Liganded ERα binds to estrogen responsive elements of target genes directly (classical) or “tethered” with Sp1 or AP1 family members to activate gene expression. The non-nuclear ERα rapid response signaling activity includes different actions as exemplified by cellular calcium mobilization, nitric oxide synthesis, and activation of signaling cascades. In order to mediate these varied activities, ERα, like all nuclear receptors, maintains the classical domain demarcations that contain different functionality, such as Activation Function -1 (AF-1), AF-2, DNA binding domain (DBD), ligand binding domain (LBD) and nuclear localization signal (NLS). Understanding the in vivo actions of these functional domains has been delineated primarily through in vitro cell culture systems. To dissect the physiological functionality we have generated different ERα mutant mouse models. Classical ERα mediated DNA responses can be abrogated by a double point mutation in the DBD (KI/KO mice) and in this mouse model, only “tethered” and rapid action responses are present, the KI/KO action corrects some effects, but does not rescue αERKO phenotypes of the uterus and mammary gland. AF-2 is the ligand activated transcription function assigned to helix 12 of the LBD. Point mutations in this region no longer allows for estradiol dependent ERα activation, but responds agonistically through AF-1 to the ERα antagonist, ICI182780 and to some SERM’s, but others remain antagonists (Raloxifen, basodioxifene) . We have generated an AF-2 mutant mouse (AF2ER mouse) to dissect the in vivo physiological functions of AF-1 and AF-2. The AF2ER mouse shows phenotypes similar to the αERKO with male and female infertility and lack of hormonal responses consistent with the estradiol inactivity and ligand independent activation. Treatment in vivo with ICI or TAM results in differential tissue activities of the AF2ER mutant receptor suggesting that ERα using AF-1 and AF-2 in different tissues to mediate its actions. Understanding the tissue selective AF-1/AF-2 utilization should provide further insight for the potential to develop tissue selective hormonal therapeutics.