We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.