BACKGROUND. Toll-like receptor-9 (TLR9) is a cellular receptor for bacterial and vertebrate DNA.In addition to cells of the immunesystem, it is also expressed in varioushuman cancer cell lines, including prostate cancer. We demonstrated previously that synthetic TLR9 ligands induce matrix metalloproteinase-13-mediated invasion in TLR9-expressing prostate cancer cells in vitro. Other studies have suggested possible sex steroid regulation of the function of the various TLRs. The role of TLR9 in the pathophysiology of prostate or any cancer is, however, unknown. METHODS. Expression of TLR9, androgen receptor (AR), or the estrogen receptors α (ERα) and β (ERβ) were studied with immunohistochemistry in prostate cancer (n=62) and benign prostatic hyperplasia (n=45) specimens. TLR9 staining scores were compared with tumor stage, Gleason score, prostate-specific antigen (PSA) concentrations before tissue sampling and with the staining scores of AR, ERα, and ERβ. RESULTS. TLR9 expression was statistically significantly increased in prostate cancer epithelium and stroma, as compared with the same cellular compartments in benign hyperplasia. Significantly increased (P=0.04) TLR9 expression was detected in cancers with high Gleason score (>7, n=23), as compared with lower Gleason scores (≤7, n=39). No statistically significant associations were detected between TLR9 expression scores and PSA concentrations or tumor staging. Prostate adenocarcinoma cells were all positive for TLR9, AR, and ERβ but negative for ERα expression. In cancer stroma cells, increased TLR9 expression was associated with increased ERα expression. CONCLUSIONS. Expression of TLR9 is increased in prostate cancer specimens, especially in the most poorly differentiated forms. © 2010 Wiley-Liss, Inc.