Tumor-associated antigens (TAA) expressed on the surface of enzootic bovine leukemia (EBL) cells were detected and separated from sera of bovine leukemia virus (BLV)-positive cattle using monoclonal antibody-conjugated immunoaffinity matrix. Eluted fraction from these sera showed 3 polypeptides with molecular weights of 70K, 52K, and 30K daltons, and these polypeptides reacted with a monoclonal antibody against TAA. However, only 70K peptide was isolated from culture supernatant of EBL B-cell line. We also tried to examine a reversed passive hemagglutination test to develop a rapid screening system of serum TAA level, but its sensitivity was below the level of detection when EBL sera was applied directly. This is the first report on the existence of tumor antigens in sera from leukemic cattle.