Abstract The androgen receptor (AR) plays a critical role in proliferation of prostate cancer cells. However, the mechanism of its action in proliferation remains unknown. We have shown previously that an AR-antagonist, Bicalutamide (Casodex), inhibits progression of synchronized LNCaP cells from G1 to S phase suggesting AR requirement in G1 phase for the cells to enter S phase (J Cell Physiol 195:337-345). In the present study we observed that a 4-hour pulse treatment with Casodex during mid- to late-G1 phase was sufficient to decrease the ability of cells to enter S phase. This brief pulse treatment with Casodex had no noticeable affect on either AR transcriptional activity or DNA synthesis in cells that had entered S phase. These observations suggested AR involvement in regulatory events, such as the assembly of pre-replication complex (pre-RC), that occur during mid- to late-G1 and are necessary for the onset of DNA synthesis and the progression of cells from G1 to S phase. Consistent with this possibility, we observed that AR co-localized and co-immunoprecipitated with Cdc6, an essential component of pre-RC, as determined by confocal microscopy and immunoprecipitation studies, respectively. Co-localization and co-precipitation of AR and Cdc6 were seen in synchronized cells that entered S phase, but not in those that were either in G0/G1 phase or prevented from entering into S phase by Casodex treatment indicating AR role in pre-RC. AR imunoprecipitate (AR-IP) also contained cyclin E and cyclin A that play a critical role in pre-RC assembly. In addition, AR also co-localized with DNA polymerase and AR-IP prepared from nuclear extracts of exponentially growing LNCaP cells contained DNA polymerase, PCNA, and ribonucleotide reductase. AR interaction with pre-RC components and the enzymes of DNA synthesis was highly specific since several other proteins such as cyclin B (a mitotic cyclin), caspase-3 (an apoptotic enzyme), PSA (AR-target gene), or GAPDH and actin (housekeeping proteins) were not associated with AR-IP. Thus AR interaction is limited to those proteins and enzymes that are involved in the onset of DNA synthesis and, therefore, the progression of prostate cancer cells from G1 to S phase. Furthermore, sucrose-density gradient analysis revealed AR co-sedimentation with cell cycle regulatory proteins, viz., cyclins E and A, and the enzymes of DNA synthesis, viz., DNA polymerase, PCNA and ribonucleotide reductase, in nuclear extracts that were prepared from S, but not G0/G1, phase cells. Thus AR exhibits a cell cycle-dependent interaction with proteins and enzymes involved in the initiation of DNA synthesis. Together, these observations point to a novel role of AR as a structural component of DNA replication machinery to exert control over proliferation of prostate cancer cells through a mechanism that is independent of its role as a transcription factor (Supported by NIH grant DK57864 and DoD grant W81XWH-05-1-0071). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3918. doi:1538-7445.AM2012-3918