298 Background: Recent reports suggest that AR aberrations are involved in the development of metastatic castration-resistant prostate cancer and in resistance to AR-targeted therapies. These aberrations include point mutations, copy number gain, and alternatively spliced AR forms. TAS3681 has demonstrated activity as a pure AR antagonist against several AR mutations and has shown an antitumor effect in an AR-v7 positive xenograft model. In order to further characterize the properties of TAS3681, we investigated its effects in cell-based AR overexpression/stabilization models and assessed the subcellular localization of FL-AR and AR-v7 in enzalutamide (Enz) -resistant cells. Methods: The Nano-bit assay was performed to analyze dimerization of FL-AR and AR-v7 proteins. Prostate cancer (PCa) cells expressing AR-v7 were treated with TAS3681, and nuclear and cytoplasmic fractions were prepared. Then the levels of FL-AR and AR-v7 protein in each fraction were determined by western blotting. AR-overexpressing PCa cells transfected with a wild-type AR expression vector were treated with TAS3681 in the presence of androgen. AR-stabilized PCa cells were transfected with a Speckle-type POZ protein (SPOP) mutant expression vector and then were treated with TAS3681. Results: AR antagonism by TAS3681 caused dose-dependent inhibition of FL-AR homodimerization (HD) and FL-AR/AR-v7 heterodimer formation in the presence of androgen. TAS3681 had no direct inhibitory effect on HD of AR-v7. However, AR downregulation by TAS3681 reduced nuclear AR-v7 protein in Enz-resistant cells under castration conditions. In AR-overexpressing PCa cells, TAS3681 reduced the AR protein and effectively suppressed both AR transactivation and cell proliferation. In contrast to Enz, TAS3681 also effectively reduced the AR protein in AR-stabilized PCa cells harboring SPOP mutation. Conclusions: TAS3681 suppresses aberrant AR activation, including AR overexpression and expression of constitutively active nuclear localized AR-v7, via downregulation of AR/AR-v7. These findings suggest that TAS3681 has the potential to overcome resistance to current AR-targeted therapies.