Critical aspects of the biology and molecular basis for prostate malignancy remain poorly understood. To reveal fundamental differences between benign and malignant growth of prostate cells, we performed gene expression profiling of primary human prostate cancer and benign prostatic hyperplasia (BPH) using cDNA microarrays consisting of 6500 human genes. Frozen prostate specimens were processed to facilitate extraction of RNA from regions of tissue enriched in either benign or malignant epithelial cell growth within a given specimen. Gene expression in each of the 16 prostate cancer and nine BPH specimens was compared with a common reference to generate normalized measures for each gene across all of the samples. Using an analysis of complete pairwise comparisons of expression profiles among all of the samples, we observed clearly discernable patterns of overall gene expression that differentiated prostate cancer from BPH. Further analysis of the data identified 210 genes with statistically significant differences in expression between prostate cancer and BPH. These genes include many not recognized previously as differentially expressed in prostate cancer and BPH, including hepsin, which codes for a transmembrane serine protease. This study reveals for the first time that significant and widespread differences in gene expression patterns exist between benign and malignant growth of the prostate gland. Gene expression analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth and identify molecular markers for diagnostic, prognostic, and therapeutic use.