The CA125 antigen, recognized by the OC125 antibody, is a tissue-specific circulating antigen expressed in ovarian cancer. The CA125 antigen is encoded by the MUC16 gene cloned by Yin and Lloyd. The full-length gene describes a complex tethered mucin protein present primarily in a variety of gynecologic tissues, especially neoplasms. OC125 and other related antibodies react with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. These antibodies are not useful as screening tools, nor can they detect the proximal residual MUC16 protein fragment after cleavage. This has limited its diagnostic and therapeutic applications. Using synthetic peptides, we raised novel-specific antibodies to the carboxy-terminal portion of MUC16 retained by the cell proximal to the putative cleavage site. These antibodies were characterized using fluorescence-activated cell-sorting analysis, enzyme-linked immunoassay, Western blot analysis, and immunohistochemistry. Each of the selected monoclonal antibodies was reactive against recombinant GST-ΔMUC16 protein and the MUC16-transfected SKOV3 cell line. Three antibodies, 4H11, 9C9, and 4A5 antibodies showed high affinities by Western blot analysis and saturation-binding studies of transfected-SKOV3 cells and displayed antibody internalization. Immunohistochemical positivity with novel antibody 4H11 was similar to OC125 but with important differences, including diffuse positivity in lobular breast cancer and a small percentage of OC125-negative ovarian carcinomas that showed intense and diffuse 4H11. Development of such antibodies may be useful for the characterization of MUC16 biology and allow for future studies in targeted therapy and diagnostics.