BACKGROUND The free prostate-specific antigen (PSA) isoform, [-2]proPSA, has been shown to be associated with prostate cancer. The study objective was to characterize the clinical utility of serum [-2]proPSA for prostate cancer detection and assess its association with aggressive disease. METHODS From among 669 subjects in a prospective prostate cancer detection study at four National Cancer Institute Early Detection Research Network clinical validation centers, 566 were eligible. Serum PSA, free PSA, and [-2]proPSA were measured (Beckman Coulter Access 2 Analyzer). RESULTS Two hundred and forty-five (43%) of the 566 participants had prostate cancer on biopsy. At 70% specificity, the sensitivity of %[-2]proPSA ([-2]proPSA/fPSA) was 54% [95% confidence interval (CI), 48-61%; null hypothesis, 40%]. Including %[-2]proPSA in a multivariate prediction model incorporating PSA and %fPSA improved the performance (P<0.01). In the 2 to 4 ng/mL PSA range, %[-2]proPSA outperformed %fPSA (receiver operator characteristic-areas under the curve, 0.73 versus 0.61; P=0.01). At 80% sensitivity, %[-2]proPSA had significantly higher specificity (51.6%; 95% CI, 41.2-61.8%) than PSA (29.9%; 95% CI, 21.0-40.0%) and %fPSA (28.9%; 95% CI, 20.1-39.0%). In the 2 to 10 ng/mL PSA range, a multivariate model had significant improvement (area under the curve, 0.76) over individual PSA forms (P<0.01 to <0.0001). At 80% sensitivity, the specificity of %[-2]proPSA (44.9%; 95% CI, 38.4-51.5%) was significantly higher than PSA (30.8%; 95% CI, 24.9-37.1%) and relatively higher than %fPSA (34.6%; 95% CI, 28.5-41.4%). %[-2]proPSA increased with increasing Gleason score (P<0.001) and was higher in aggressive cancers (P=0.03). CONCLUSIONS In this prospective study, %[-2]proPSA showed potential clinical utility for improving prostate cancer detection and was related to the risk of aggressive disease. IMPACT The addition of %[-2]proPSA could affect the early detection of prostate cancer.