Abstract Background: The percentage of free prostate-specific antigen (%fPSA) has been shown to improve specificity for the diagnosis of prostate cancer (PCa) over total PSA (tPSA). A multicenter study was performed to evaluate the diagnostic value of a %fPSA-based artificial neural network (ANN) in men with tPSA concentrations between 2 and 20 μg/L for detecting patients with increased risk of a positive prostate biopsy for cancer. Methods: We enrolled 1188 men from six different hospitals with PCa or benign prostates between 1996 and 2001. We used a newly developed ANN with input data of tPSA, %fPSA, patient age, prostate volume, and digital rectal examination (DRE) status to calculate the risk for the presence of PCa within different tPSA ranges (2–4, 4.1–10, 2–10, 10.1–20, and 2–20 μg/L) at the 90% and 95% specificity or sensitivity cutoffs, depending on the tPSA concentration. ROC analysis and cutoff calculations were used to estimate the diagnostic improvement of the ANN compared with %fPSA alone. Results: In the low tPSA range (2–4 μg/L), the ANN detected 72% and 65% of cancers at specificities of 90% or 95%, respectively. At 4–10 μg/L tPSA, the ANN detected 90% and 95% of cancers with specificities of 62% and 41%, respectively. Use of the ANN with 2–10 μg/L tPSA enhanced the specificity of %fPSA by 20–22%, thus reducing the number of unnecessary biopsies. Conclusions: Enhanced accuracy of PCa detection over that obtained using %fPSA alone can be achieved with a %fPSA-based ANN that also includes clinical information from DRE and prostate volume measurements.