Background: Prostate cancer antigen 3 (PCA3) is considered to be prostate cancer (PCa) specific and highly overexpressed in cancer. Therefore a high PCA3 score should result in a high positive predictive value (PPV) and specificity for a positive biopsy. Objective: Our aim was to reevaluate, retest PCA3, and rebiopsy men with an initial PCA3 >= 100 and no PCa detected and compare the results with a random cohort of men with an initial PCA3 < 100. Design, setting, and participants: We invited men 63-75 yr of age with a PCA3 >= 100 for retesting and a control group with an initial PCA3 < 100 to participate in the European Randomized Study of Screening for Prostate Cancer, section Rotterdam. Interventions: Blood and urine sampling were used to determine prostate-specific antigen (PSA) and PCA3. Prostate biopsies were performed if the PSA was >= 2.5 ng/ml and/or the PCA3 score was >= 35. Measurements: We correlated the initial and reevaluated PCA3 scores. Our assessment of the PPV after rebiopsy was based on the newly determined PCA3 score. Results and limitations: After a mean study period of 19 mo, more cases of PCa were detected in rebiopsied men with initial PCA3 scores >= 100 than in the controls with PCA3 scores < 100 (30.0% vs 18.8%). Combining initial and rebiopsy data resulted in a PPV of 52.2% in men with PCA3 >= 100. Over time, changes in PSA and PCA3 levels were quite different. Conclusions: In spite of our rescreened population, PPV and specificity were comparable with all reported studies of men with PCA3 scores >= 100. These findings do not explain why these PCA3 scores were excessively high in spite of the absence of biopsy-detectable PCa. (C) 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved.