PURPOSE: Most men with increased serum prostate specific antigen and negative biopsy require repeat biopsy because of the lack of a sensitive and specific prostate cancer detection test. In this study we evaluated the diagnostic potential of a duplex assay for prostate cancer by quantifying transcript levels of alpha-methylacyl-CoA racemase and prostate cancer antigen 3 in urine sediments following prostatic massage. MATERIALS AND METHODS: Urine sediments from 92 patients, 43 with and 49 without prostate cancer, were collected after digital rectal examination. Transcript levels of AMACR, PCA3 and PSA in total RNA isolated from these samples were determined by absolute quantitative real-time polymerase chain reaction. AMACR and PCA3 scores were obtained by normalizing the transcript level to that of prostate specific antigen for each sample and multiplying by 100. RESULTS: AMACR (p = 0.006) and PCA3 (p = 0.014) scores, but not serum prostate specific antigen (p = 0.306), distinguished specimens from patients with prostate cancer from specimens from patients without prostate cancer, and ROC analysis established the diagnostic cutoff scores for the AMACR and PCA3 tests at 10.7 and 19.9, respectively. As determined from these cutoff scores the AMACR test had 70% (95% CI 56-83) sensitivity and 71% (95% CI 59-84) specificity, whereas the PCA3 test had 72% (95% CI 59-85) sensitivity and 59% (95% CI 45-73) specificity for prostate cancer detection. The combined use of AMACR and PCA3 scores in a dual marker test increased sensitivity to 81% (95% CI 70-93) and specificity to 84% (95% CI 73-94). CONCLUSIONS: Urinary AMACR and PCA3 tests were superior to a serum prostate specific antigen test for detecting prostate cancer. Their combined use in a dual marker test further improved sensitivity and accuracy, and could serve as a surveillance test after repeat negative prostate biopsies.