Genomic rearrangements involving genes encoding erythroblast transformation-specific transcription factors are commonly present in prostate cancer. The TMPRSS2-ERG gene fusion that leads to ERG overexpression occurs in ~70% of prostate cancers. Implementation of fusion gene detection in pathological practice, however, has been hampered by the lack of reliable ERG antibodies. The objective of this study was first to compare ERG immunohistochemistry using the recently described antibody EPR3864 with ERG mRNA by quantitative PCR and, second, to investigate ERG immunohistochemistry in diagnostic prostate cancer needle biopsies. We analyzed 41 primary prostate adenocarcinomas obtained by radical prostatectomy and 83 consecutive prostate cancer needle biopsies. In the prostatectomy specimens, immunohistochemical ERG expression was highly concordant with the ERG mRNA overexpression (sensitivity 100% and specificity 85%). ERG overexpression was due to TMPRSS2-ERG gene fusion in all cases. ERG protein expression was identified in 51/83 adenocarcinomas (61%) on needle biopsies. ERG expression was more frequent in tumors infiltrating ≥2 needle biopsies (P<0.001) or occupying ≥50% of a single biopsy (P=0.018). Expression of ERG also occurred in 11/21 (52%) high-grade prostate intraepithelial neoplasia lesions. In 5/87 (6%) needle biopsies containing benign secretory glands, weak ERG staining was focally observed. In all of these cases, respective glands were adjacent to adenocarcinomas. In conclusion, immunohistochemistry for ERG strongly correlated with ERG mRNA overexpression and was specific for prostate cancer on needle biopsies. Therefore, ERG immunohistochemistry is an important adjunctive tool for pathophysiological studies on ERG gene fusions, and might support the pathological diagnosis of adenocarcinoma in a subset of prostate needle biopsies.