Antigenic epitopes of nonspecific cross-reacting antigen (NCA) recognized by 8 different monoclonal antibodies (MAbs) were analyzed in relation to the domain structures of NCA [domains N, I (A1-B1) and M] and CEA [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed cDNAs for NCA-N, NCA-N-I-M, CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M in a eukaryotic expression vector, pdKCR-dhfr, and expressed them in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested against eight different MAbs reactive with NCA. All 8 MAbs had been shown to recognize the protein epitopes of the NCA molecule and classified into two groups in terms of the reactivity with NCA and CEA; Group X, 5 clones reactive with both NCA and CEA; and Group Y, 3 clones reactive only with NCA. The epitopes recognized by two of five Group X MAbs were found to be present on the domain N of the NCA molecule as well as of the CEA molecule, and those of the three others were on the domain I (A1-B1) of both molecules, respectively. All three epitopes of Group Y MAbs, which were unique to NCA, were present on the domain I (A1-B1) but not on the domain N of the NCA molecule. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the NCA molecule and may be useful to study the functions of the NCA molecule, especially those of the respective domains.