ABSTRACT: We analyzed ten monoclonal antibodies and three polyclonal antisera directed against human chorionic gonadotropin by immunoassay in order to determine which pairs of antibodies are capable of binding simultaneously to hCG. A relative orientation of epitopes on the surface of hCG could be inferred from a matrix of data describing the abilities of particular antibody pairs to inhibit competitively or enhance the binding of each other. These results indicated that the binding site of each of the antibodies could be assigned to one of three different regions of the hCG molecule, and at least one antibody binding within each region exhibited a substantial preference for hCG relative to human luteinizing hormone. One of these regions is the COOH-terminal portion of the hCGβ subunit. A second region contains the binding site for the SB6 rabbit antiserum (which has been widely employed in clinical studies) as well as the binding sites of several monoclonal antibodies. Antibodies to this second region characteristically bind both the whole hormone and the free β subunit. A third region contains the binding sites of a previously reported human antiserum and also several monoclonal antibodies. These antibodies characteristically react preferentially with the intact hormone relative to the free subunits. The information contained in the type of epitope map described here can be used as the rational basis for the design of two-site immunoradiometric assays for hCG and/or its subunits.