Epithelial cell adhesion molecule (Ep-CAM) is a 40kDa transmembrane glycoprotein overexpressed in majority of tumor epithelial cells and has a major morphoregulatory function, relevant not only to epithelial tissue development, but also in carcinogenesis and tumor progression. Since Ep-CAM localizes at the cell surface of most carcinomas, the molecule is an attractive target for immunotherapy and several strategies have been deployed to treat cancer using Ep-CAM targeting, including MAb therapy. For improving effective targeting of this protein for diagnostics in various clinical samples, we generated and characterized an anti-Ep-CAM MAb (C4) using recombinant Ep-CAM protein, comprising the highly immunogenic domain. The specificity of C4-MAb was characterized in Ep-CAM positive cell lines (PC3 and MCF-7) by flow cytometry and immunofluorescence. The immunohistochemistry analysis in clinical tissue samples showed specific detection of epithelial antigens in breast, colon, stomach, and prostate carcinomas. Thus, this Ep-CAM MAb (C4-MAb) could be used for both diagnostic and therapeutic applications due to its specificity. © Mary Ann Liebert, Inc.