Abstract Hypoxia is an important microenvironmental factor influencing tumor progression and treatment response. The understanding how hypoxia is regulating the behavior of cancer cells has to be further explored. The androgen receptor (AR) acts as a transcriptional factor controlling development or progression of prostate cancer (PC). Androgen ablation by castration is the common therapy for advanced PC but eventually most of the tumors relapse to a castration-resistant state and grow even in a low androgen environment. The remaining androgens that are still present after therapy may continue to promote AR activation. The most potent AR ligands, testosterone (T) and dihydrotestosterone (DHT), can be synthesized locally in the prostate from adrenal androgens and castration-resistant PC and bone metastases are shown to express increased levels of enzymes responsible for androgen metabolism (Akr1c3 one of several enzymes). As castration results in a transient tissue hypoxia and solid tumors are generally hypoxic prior to treatment we investigated how hypoxia affects AR activation in androgen-sensitive prostate cancer CWR22Pc cells additional to steroidal treatment. Hypoxia upregulated the mRNA expression of the androgen-responsive NKX3.1 and prostate specific antigen (PSA), and facilitated translocation of AR to the nucleus in the presence of 0,01 nM DHT or 100 nM androstenediol. The androstenediol activation of AR simultaneously increased mRNA expression of the androgen converting enzyme Akr1c3 possibly resulting in metabolism of androstenediol to T or DHT. This study shows that the AR can be hypersensitized to low levels of androgens in PC cells grown in a hypoxic milieu as in solid tumors and after castration. A consequence of exposure of PC cells to hypoxia is a possible increase in local biosynthesis of T and DHT from adrenal androgens or a direct ligand binding effect by adrenal androgen to activate the AR. Citation Format: Stina Rudolfsson, Erik Toolanen, Anders Bergh. Hypoxia enhances androgen receptor activation in prostate cancer cells in response to low levels of androgens [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B8.