In this report, we describe the application of a systematic, genome-based approach to identify prostein, a novel prostate-specific protein expressed in normal and malignant prostate tissues. Characterization of the prostein gene shows that prostein cDNA encodes a 553-amino acid protein. The protein is predicted to be a type IIIa plasma membrane protein with a cleavable signal peptide and 11 transmembrane-spanning regions. The prostein gene is located on chromosome 1 at the WI-9641 locus between q32 and q42. Prostein mRNA is shown to be uniquely expressed in normal and cancerous prostate tissues using Northern blot, eDNA microarray, and real-time PCR analyses. Furthermore, prostein mRNA expression does not appear to be prostate tumor grade related and is restricted exclusively to prostate cell lines. Immunohistochemical staining using a mouse monoclonal antibody generated against prostein demonstrates that this protein is specifically detected in prostate tissues both at the plasma membrane and in the cytoplasm. Prostein expression is androgen responsive because treatment of LNCaP cells with androgen up-regulates prostein message and protein expression levels. These results validate prostein as a prostate-specific marker with potential utility in the diagnosis and treatment of prostate cancer.