Abstract The Androgen Receptor (AR) is a ligand inducible transcription factor that drives expression of genes contributing to the growth, recurrence and metastasis of prostate cancer (PCa). The AR is activated by steroid hormones (androgens) that bind to a C-terminal ligand-binding domain (LBD) and cause the transcription factor to enter the nucleus, dimerize and interact with DNA via its conserved DNA-binding domain (DBD). Accordingly, treatment for PCa involves reducing DHT production or using small molecules (i.e. antiandrogens) to compete with the specific interaction between hormones and the AR-LBD, preventing AR activation. This treatment will eventually fail as recurrent PCa changes into a castration resistant form (CRPC) where AR signalling continues even in the absence of androgens. The occurrence of CRPC is driven, in part, by mutations in the AR-LBD that alter the binding of antiandrogens, or by the presence of constitutively active AR splice variants that lack the LBD, effectively removing the site of action of conventional antiandrogens. Here, we describe a surface exposed pocket on the AR-DBD, based on its 3D-crystal structure, which may qualify as an alternative target for small molecule inhibitors of AR transcriptional activity. Following virtual screening of many compounds, we characterize the interaction of the AR-DBD with various potential inhibitors and measure the efficacy of these agents to block the transcriptional activity of both full-length and splice variant AR in cell culture. Drug-inhibition was reduced upon site-directed mutagenesis of amino-acid residues in the DBD that are suspected of interacting with the compounds. Biochemical approaches are used to further examine the physical interaction of the purified AR-DBD protein with small molecules and to investigate their effect on protein-DNA interactions in vitro. Lastly, we use confocal microscopy of live PCa cells to investigate the effect of our compounds on the spatial localization and dimerization of fluorescently tagged AR proteins. These results illustrate the feasibility of targeting the AR-DBD to potentially inhibit the reactivation of AR transcriptional activity that is observed in CRPC. Citation Format: Kush Dalal, Huifang Li, Mani R. Moniri, Fuqiang Ban, Aishwariya Sharma, Artem Cherkasov, Paul S. Rennie. Targeting the DNA binding domain of the androgen receptor as a potential therapy for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2014-1235