We evaluated the usefulness of double immunohistochemical staining for cytokeratin (CK)5/6 and alpha-methylacyl coenzyme A racemase (AMACR) applied sequentially on 1 slide by assessing 223 foci in 110 consecutive prostate specimens. Double-chromogen reaction was used to visualize the antibodies: brown for CK5/6 and red for AMACR. Staining was scored as diffuse, focal, or negative. To establish the diagnosis, CK5/6 and AMACR were correlated with the morphologic features. All cancers lacked CK5/6 staining (100% specificity). AMACR showed diffuse or focal positivity in cancer, high-grade prostatic intraepithelial neoplasia, and atypia in 96.8% (120/124), 85% (22/26), and 80% (16/20) of cases, respectively. In atypical cases, diagnosis was because of non-immunohistochemical staining reasons in 80% of cases. In adenosis (n = 14), AMACR was diffusely positive in 4 cases (29%). Double immunohistochemical staining for CK5/6 and AMACR is a simple assay to perform and may be used as an alternative to antibody cocktails for routine evaluation of problematic prostate specimens.