The androgen receptor (AR) is involved in the development and progression of prostate cancers. However, the mechanisms by which this occurs remain incompletely understood. In previous reports, hepatocyte nuclear factor-3 alpha (HNF-3 alpha) has been shown to be expressed in the epithelia of the prostate gland, and has been determined to regulate the transcription of prostate-specific genes. In this study, we report that HNF-3 alpha functions as a novel corepressor of AR in prostatic cells. HNF-3 alpha represses AR transactivation on target promoters containing the androgen response element (ARE) in a dose-dependent manner. HNF-3a interacts physically with AR, and negatively regulates AR transactivation via competition with AR coactivators, including GRIP1. Furthermore, HNF-3 alpha overexpression reduces the androgen-induced expression of prostate-specific antigen (PSA) in LNCaP cells. Taken together, our findings indicate that HNF-3 alpha is a novel corepressor of AR, and predict its effects on the proliferation of prostate cancer cells. (c) 2007 Elsevier Inc. All rights reserved.