Background/Aim: To evaluate cancer/testis (CT) antigens as targets for immunotherapy or vaccine approaches in prostate cancer. PATIENTS AND METHODS: We investigated the antibody response in 181 patients with prostate cancer, 83 benign prostate hyperplasia (BPH) patients, and 39 healthy donors against 13 different CT antigens recombinantly expressed on yeast surface (RAYS) and compared the results to antigen expression in tumor tissue. We then used the yeast clone expressing the most promising antigen directly as a vaccine to elicit potent cellular immunity. RESULTS: The antibody response to NY-ESO-1 was more frequent (20%) and strong compared to other investigated antigens, and was associated with progressive disease. Interestingly, it was also detected in several BPH patients (9%). Feeding dendritic cells with NY-ESO-1-expressing yeast cells resulted in efficient HLA presentation and activation of specific CD3(+) T-cells. CONCLUSION: The RAYS approach offers a fast means of analyzing serological autoreacitvity in cancer patients and serves as an effective anticancer vaccine platform.