Cancer antigen 125 (CA-125) is the most widely used tumor marker for ovarian cancer. Thus, monoclonal antibodies (MAbs) against CA-125 are valuable reagents for the development of diagnostic tests and immunotherapy. We describe here the generation and characterization of three novel hybridoma cell lines producing MAbs against CA-125. CA-125 purified from culture supernatant of ovarian carcinoma cell line OVCAR-3 by affinity chromatography, was used for immunization of BALB/c mice. Three stable cell lines (3C8, 2B6, and 5A12) were selected for production of antibodies against CA-125 and were expanded in mass culture. All three antibodies were shown to recognize linear epitopes. Antibodies 2B6 and 5A12 were determined to recognize epitope cluster B (M 11-like); MAb 3C8 was classified as group A-epitope binders (OC 125-like). The antibodies produced may be used for the development and improvement of CA-125 immunoassays.