Breast tumor kinase (BRK) is an intracellular tyrosine kinase expressed in differentiating epithelial cells of the gastrointestinal tract and skin, and in several epithelial cancers including carcinomas of the breast and colon. We examined expression of BRK and its mouse ortholog Src-related intestinal kinase (Sik) in prostate tissues and detected it in the nuclei of normal luminal prostate epithelial cells. BRK localization was then examined in 58 human prostate biopsy samples representing various grades of prostate cancer. While nuclear localization of BRK was present in well-differentiated tumors, it was absent in poorly differentiated tumors. However localization of Sam68, a nuclear substrate of BRK/Sik, was unaltered in all prostate tumors examined. Consistent with these results, nuclear BRK was detected in the more differentiated androgen-responsive LNCaP human prostate cancer cell line that is poorly tumorigenic in host animals, but it was primarily cytoplasmic in the undifferentiated androgen-unresponsive PC3 prostate cancer cell line that forms aggressive tumors. While PC3 cells expressed higher levels of endogenous BRK than LNCaP cells, BRK was less active in these cells. Our data suggest that BRK plays a role in differentiation of prostate epithelial cells. Altered BRK localization and/or activity may provide a prognostic indicator for prostate tumor progression and be a potential target for therapeutic intervention.