The expression of the prostate-specific membrane antigen (PSMA) glycoprotein recognized by the murine monoclonal antibody (MAb) 7E11-C5.3 has been shown to be highly restricted to prostate epithelium. Although the conjugated form of this MAb (CYT-356) may soon be used clinically for in vivo imaging of extraprostatic disease, few details regarding the nature of the antigenic epitope of PSMA have been reported. This study was carried out to analyze the MAb 7E11-C5.3 epitope on PSMA using standard biochemical techniques, and the antigenic epitope was mapped with synthetic peptides. The MAb 7E11-C5.3 epitope was susceptible to both periodic acid oxidation and proteolytic digestion, which indicated that the antigen consisted of a glycoprotein. However, additional biochemical assays such as sodium borohydride, tunicamycin treatment, and digestion with glycosidases failed to abrogate MAb 7E11-C5.3 binding. Epitope mapping with synthetic peptides demonstrated the epitope to be localized to the intracellular domain at the N-terminus of the PSMA molecule with a minimal reactive peptide consisting of six amino acids (MWNLLH). The synthetic peptides were treated with periodic acid, which resulted in inhibition of antibody binding, suggesting that treatment of the PSMA antigen resulted in damage to the peptide chain. These data suggest that the MAb 7E11-C5.3 does not recognize a glycopeptide as was initially thought, but recognizes an intracellular epitope consisting of only the primary polypeptide chain. Further studies are needed to determine how CYT-356 is able to image tumors in vivo when the antigenic epitope is intracellular.