Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5′-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction. (Cancer Res 2006; 66(21): 10594-602)