To identify tumor‐associated antigens as putative targets for developing immunotherapies against prostate cancer, we investigated the ability of T cells derived from the peripheral blood lymphocytes of prostate cancer patients to recognize autologous tumor cells. The technical challenge of growing in vitro carcinoma cell lines from small prostate cancer samples was previously addressed by immortalization of early epithelial cell cultures with the HPV16 transforming proteins E6 and E7 and by genetic characterization of the carcinoma and normal prostate cell lines. In our study, peripheral blood lymphocytes were stimulated in vitro using autologous IFNγ‐treated prostate carcinoma cells transduced with the B7.1 molecule as a source of T‐cell costimulation. Tumor‐specific CD8+ T lymphocytes were obtained from 3 of 6 prostate cancer patients tested and included T cells restricted by classical and nonclassical HLA molecules. In 1 case, we demonstrated that the prostate cancer‐reactive T cells were TCRα/β+ and recognized autologous tumor cells but not autologous normal cells in the context of HLA‐B or ‐C molecules. These results validate the approach of in vitro stimulation of peripheral blood lymphocytes from prostate cancer patients with autologous tumor cell lines to isolate prostate cancer‐specific T cells and demonstrate the existence of a functionally diverse immune response against prostate cancer. Published 2001 Wiley‐Liss, Inc.