Abstract Prostate cancer is the most common malignancy affecting men. One diagnostic problem is detection of small foci of prostate cancer in biopsy specimens. Currently, clinical diagnosis of prostate cancer involves an H&E histopathologic examination of needle biopsies and immunohistochemical (IHC) evaluation of the presence/absence of tissue α-methylacyl coenzyme A racemase (AMACR, p504s) and basal cell markers [p63, high molecular weight (HMW) cytokeratins (CKs)]. The goal of this study was to evaluate the performance of an IHC assay using the novel anti-p504s (clone SP116) rabbit monoclonal antibody and compare it with the performance of the commercially available antibody clone 13H4. Prostate tissues from 207 patients (145 prostate cancers and 62 nonmalignant prostates) were evaluated for p504s expression using IHC with the anti-p504s (clone SP116) rabbit monoclonal antibody and the anti-p504s (clone 13H4) rabbit monoclonal antibody, respectively. Tissues were stained on the BenchMark ULTRA instruments with ultraView Red detection kit, using either the newly developed protocol optimized for SP116 clone or the optimized protocol recommended by NordiQC for 13H4 clone. In addition, the same tissues were also stained with either the anti-p504s (SP116) antibody and VENTANA Basal Cell Cocktail (34βE12+p63) using ultraView DAB/ultraView Red dual stain procedure on a BenchMark ULTRA instrument, or with Biocare antibodies cocktail [CK HMW+p63+AMACR (RM)] on the Biocare IntelliPath staining instrument using Biocare detection according to manufacturer’s protocol. The 13H4 clone is used in this cocktail to detect the p504s expression. The prostate cancer tissues evaluated with the IHC assays using anti-p504s antibodies showed moderate/strong cytoplasmic staining, while nonmalignant tissues showed weak/focal or negative staining. The reproducibility and repeatability of the newly developed IHC assay using anti-p504s (SP116) antibody was excellent. The sensitivities to detect prostate cancer were similar for both clones [89% (123/139) for clone SP116, 91% (126/139) for clone 13H4] when a single assay was used; however, the specificity to detect prostate cancer cells was 89% (54/61) for the new SP116 clone and 56% (34/61) for clone 13H4. Using the dual assay, the expected staining pattern (p504s -positive/basal cell markers-negative) was observed in 91% (123/135) prostate cancers stained with the new SP116 clone of anti-p504s antibody and basal cell cocktail, while this pattern was observed in 85% (111/131) of cancer cases stained with the Biocare cocktail. In summary, the data from this study indicate that the SP116 clone exhibits similar sensitivity but improved specificity compared to 13H4 clone in detecting malignant prostate glands when used in a single assay on the BenchMark ULTRA instrument. However, the performance of these two clones is similar when used together with basal cell markers in the dual IHC assays. Citation Format: Ryan Miller, Carol Jones, Matthew Buckwalter, Katerina Dvorak. Immunohistochemistry using anti-p504s antibody in prostate cancer: Comparison of novel and on-market antibody clones [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A086.