The aim of this study was to identify and validate differentially expressed genes in matched pairs of benign and malignant prostate tissue. Samples included 29 histologically verified primary tumors and 23 benign controls. Microarray analysis was initially performed using a sequence verified set of 40,000 human cDNA clones. Among the genes most consistently and highly upregulated in prostate cancer was the ETS family transcription factor ERG (ETS related gene). This finding was validated in an expanded patient series (37 tumors and 38 benign samples) using DNA oligonucleotide microarray and real-time quantitative PCR assays. ERG was 20- to more than 100-fold overexpressed in prostate cancer compared with benign prostate tissue in more than 50% of patients according to quantitative PCR. Surprisingly, ERG mRNA levels were found to be significantly higher in the endothelial cell line, HUVEC, than in the prostate cell lines PC3, DU145 and LNCaP. In situ hybridization of prostate cancer tissue revealed that ERG was abundantly expressed in both prostate cancer cells and associated endothelial cells. The consistency and magnitude of ERG overexpression in prostate cancer appeared unique, but several related ETS transcription factors were also overexpressed in matched pairs of tumor and benign samples, whereas ETS2 was significantly underexpressed. Our findings support the hypothesis that ERG overexpression and related ETS transcription factors are important for early prostate carcinogenesis.