Prostate-specific membrane antigen (PSMA) is a trans-membrane glycoprotein recognized by the murine monoclonal antibody (MAb) 7EII-C5.3 both in its native (CYT-351) and immunoconjugate form (CYT-356). Previous studies have shown that tissue expression of PSMA is highly restricted to prostate tissues. In this study, a definitive immunohistochemistry evaluation was performed to assess PSMA expression in prostate tissues. A stain index was established by multiplying the percentage of stained cells by the intensity of the stained cells to provide a quantitative measurement of PSMA expression in the various tissue types. The cellular location of PSMA, its correlation with clinical status, and its comparison with the expression of prostate-specific antigen (PSA) were evaluated. Prostate-specific membrane antigen was found to be highly expressed in most of the normal intraepithelial neoplasia, and the primary and metastatic prostate tumor specimens evaluated. In contrast to PSA, PSMA expression was often heterogeneous with variable staining patterns, ranging from a low-level diffuse cytoplasmic staining in normal prostate epithelium to very intense cytoplasmic and focal membrane staining in high-grade primary carcinomas and metastatic tissues. The predominant cytoplasmic staining was expected because the antigenic epitope of the PSMA transmembrane glycoprotein recognized by MAb 7EII-C5.3 is located in the cytoplasmic domain. Benign prostate tumors, ie, hypertrophy, showed the lowest expression of PSMA with a stain index of 52, compared with stain indexes of 146 and 258 for normal prostate and bone metastatic tissues, respectively. The reason for the apparent down-regulation of PSMA in benign prostate tissue is unknown but may be related to a splicing variant or post-translational modification of PSMA. Expression of PSMA was observed to increase with increasing pathologic grade, but not with clinical stage. Although PSMA was overexpressed in poorly differentiated and metastatic prostate tumors, expression in the primary tumor did not correlate with nodal status, extracapsular penetration, or seminal vesicle invasion. These results suggest that PSMA is not a useful biomarker of disease progression; however, high expression does appear to be associated with the more aggressive prostate carcinoma phenotype. The restricted specificity, differential prostate tissue expression, and overexpression of PSMA in metastatic tissues support the continued study of this unique prostate tumor-associated biomarker for developing new strategies for diagnostic and therapy of prostate cancer.