Context: Molecular profiling of prostate cancer has led to the identification of candidate biomarkers and regulatory genes. Discoveries from these genome-scale approaches may have applicability in the analysis of diagnostic prostate specimens. Objectives: To determine the expression and clinical utility of α-methylacyl coenzyme A racemase (AMACR), a gene identified as being overexpressed in prostate cancer by global profiling strategies. Design: Four gene expression data sets from independent DNA microarray analyses were examined to identify genes expressed in prostate cancer (n=128 specimens). A lead candidate gene, AMACR, was validated at the transcript level by reverse transcriptase polymerase chain reaction (RT-PCR) and at the protein level by immunoblot and immunohistochemical analysis. AMACR levels were examined using prostate cancer tissue microarrays in 342 samples representing different stages of prostate cancer progression. Protein expression was characterized as negative (score=1), weak (2), moderate (3), or strong (4). Clinical utility of AMACR was evaluated using 94 prostate needle biopsy specimens. Main Outcome Measures: Messenger RNA transcript and protein levels of AMACR; sensitivity and specificity of AMACR as a tissue biomarker for prostate cancer in needle biopsy specimens. Results: Three of 4 independent DNA microarray analyses (n=128 specimens) revealed significant overexpression of AMACR in prostate cancer (P<.001). AMACR up-regulation in prostate cancer was confirmed by both RT-PCR and immunoblot analysis. Immunohistochemical analysis demonstrated an increased expression of AMACR in malignant prostate epithelia relative to benign epithelia. Tissue microarrays to assess AMACR expression in specimens consisting of benign prostate (n=108 samples), atrophic prostate (n=26), prostatic intraepithelial neoplasia (n=75), localized prostate cancer (n=116), and metastatic prostate cancer (n=17) demonstrated mean AMACR protein staining intensity of 1.31 (95% confidence interval, 1.23-1.40), 2.33 (95% Cl, 2.13-2.52), 2.67 (95% Cl, 2.52-2.81), 3.20 (95% Cl, 3.10-3.28), and 2.50 (95% Cl, 2.20-2.80), respectively (P<.001). Pairwise comparisons demonstrated significant differences in staining intensity between clinically localized prostate cancer compared with benign prostate tissue, with mean expression scores of 3.2 and 1.3, respectively (mean difference, 1.9; 95% Cl, 1.7-2.1; P<.001). Using moderate or strong staining intensity as positive (score=3 or 4), evaluation of AMACR protein expression in 94 prostate needle biopsy specimens demonstrated 97% sensitivity and 100% specificity for detecting prostate cancer. Conclusions: AMACR was shown to be overexpressed in prostate cancer using independent experimental methods and prostate cancer specimens. AMACR may be useful in the interpretation of prostate needle biopsy specimens that are diagnostically challenging.