High molecular mass glycoproteins detected by the monoclonal antibody OC125 were found in secreted and solubilized materials from the ovarian cancer cell line, HOC-I. The CA125 antigen has been isolated from HOC-I cell extract and conditioned media by perchloric acid precipitation, gel filtration, and OC125 affinity purification. The higher molecular mass complexes (molecular masses were estimated to be > 1,000KDa) was predominantly found in extracts of cells grown in vitro or in ovarian cancer tissues, whereas the lower molecular mass antigen (molecular masses were estimated to be 110-400KDa) was the major component in conditioned media and sera from patients with ovarian cancer. Double determinant enzyme-linked immunosorbent assays were carried out with several antibodies (OC125, NS19-9, TKH-2, B72.3, CC49, MA54, MA61, PC47H) immobilized on a solid phase to bind different epitopes on antigens in clinical samples. Biotinylated OC125 was used as a probe to detect the CA125 antigen that had been bound via different epitopes. When TKH-2 was used to bind the antigen and OC125 used as a probe, the coexpression of CA125/sialyl Tn was observed in 12(22%) of 55 sera from patients with ovarian cancer. In some cases, sialyl Tn or CA19-9 antigens may be present on the CA125 macromolecular complexes.