Eleven hybridoma clones producing monoclonal antibodies to carcinoembryonic antigen (CEA) were prepared by fusions of the mouse myeloma cell line P3-X63-Ag8-U1 with splenocytes of BALB/c mice immunized with highly purified CEA. Their specificities were systematically analyzed by radioimmunoassay for reactivity with CEA and four related normal antigens: nonspecific cross-reacting antigen (NCA), nonspecific cross-reacting antigen-2 (NCA-2), normal fecal antigen-1 (NFA-1), and normal fecal antigen-2 (NFA-2). Antibody-antigen profiles revealed four groups of hybridomas. The Group I antibodies from two clones reacted with sites shared among CEA, NCA, NCA-2, and NFA-2. The Group II antibody from one clone recognized an epitope common to CEA, NCA-2, and NFA-2. The Group III antibodies from five clones reacted with sites commonly residing on CEA, NCA-2, NFA-1, and NFA-2, whereas antibodies of Group IV from three clones bound only CEA. Mutual inhibition assays between antibodies in the respective groups for CEA binding further revealed that at least eight different epitopes can be identified on the CEA molecule: two in Group I, one in Group II, three in Group III, and two in Group IV, respectively. Three monoclonal antibodies in Group IV appeared to be specific for CEA, but they did not react with any of five purified CEA preparations other than that used for immunization. In 13 sera from 50 various cancer patients with elevated CEA levels, however, we detected the CEA molecule reactive with these antibodies, indicating that the epitopes recognized by the Group IV antibodies may be of allotypic characters. These epitopes were unrelated to main blood group antigens and did not show any organ specificity. All of the eight epitopes identified in this study were found to be resistant to neuraminidase, mixed glycosidases, and Smith degradation (SI-stage) as determined by a competitive radioimmunoassay, suggesting that they reside on the protein moiety of the CEA molecule. Six epitopes were sensitive to reduction and alkylation, but two other epitopes (one in Group II and the other in Group III) were resistant to these treatments. This result indicates that the latter two epitopes are conformation-independent.