Abstract Background The Prostate CAncer gene 3 ( PCA3 ) assay has shown promise as an aid in prostate cancer (pCA) diagnosis in identifying men with a high probability of a positive (repeat) biopsy. Objective This study evaluated the clinical utility of the PROGENSA PCA3 assay. Design, setting, and participants This European prospective, multicentre study enrolled men with one or two negative biopsies scheduled for repeat biopsy. Measurements After digital rectal examination (DRE), first-catch urine was collected to measure PCA3 mRNA concentration and to calculate the PCA3 score. The PCA3 score was compared to biopsy outcome. The diagnostic accuracy of the PCA3 assay was compared to percent of free prostate-specific antigen (%fPSA). Results and limitations In 463 men, the positive repeat biopsy rate was 28%. The higher the PCA3 score, the greater the probability of a positive repeat biopsy. The PCA3 score (cut-off of 35) had a greater diagnostic accuracy than %fPSA (cut-off of 25%). The PCA3 score was independent of the number of previous biopsies, age, prostate volume, and total prostate-specific antigen (PSA) level. Moreover, the PCA3 score was significantly higher in men with high-grade prostate intraepithelial neoplasia (HGPIN) versus those without HGPIN, clinical stage T2 versus T1, Gleason score ≥7 versus <7, and “significant” versus “indolent” (clinical stage T1c, PSA density PSAD <0.15 ng/ml, Gleason score in biopsy ≤6, and percent positive cores ≤33%) pCA. Conclusions The probability of a positive repeat biopsy increases with rising PCA3 scores. The PCA3 score was superior to %fPSA for predicting repeat prostate biopsy outcome and may be indicative of clinical stage and significance of pCa.