Carcinoembryonic antigen (CEA), a glycosylated protein of M(r) 180 kDa, is one of the most widely used human tumor markers. A majority of gastrointestinal cancers as well as breast and nonsmall cell lung carcinomas express CEA. We have previously described a recombinant baculovirus BVCEA-140 expressing the full-length human CEA and a variant, BVCEA-16, that encodes only the NH2-terminal domain, as well as a recombinant (BVNCA) expressing the closely related molecule nonspecific cross-reactive antigen (NCA). We have now compared a panel of 24 anti-CEA and anti-NCA monoclonal antibodies (MAbs) for their ability to bind to these recombinant CEA and NCA proteins, as well as with a new 60 kDa subgenomic form designated BVCEA-60. The epitope mapping studies indicate that all the CEA specific MAbs can recognize BVCEA-140. We also compared the sugar composition of BVCEA-140 to native CEA, using a lectin-linked immunoradiometric assay. The results demonstrated that both the native and recombinant baculovirus CEA contain simple high-mannose carbohydrates as well as biantennary and biantennary hybrid complexes. However, native CEA also contains triantennary and tetraantennary complex sugars, while the recombinant CEA molecule does not. Immunogenicity of the recombinant CEA molecules was demonstrated in mice. ELISA and Western blot analyses were used to determine the cross-reactivity of the anti-CEA sera. Mice immunized with BVCEA-140 elicit antibodies that are reactive to native CEA. When the BVCEA-16 was used as an immunogen, the antisera failed to detect native CEA or BVCEA-140. These studies demonstrate that minor sugar differences exist between native and baculovirus-derived CEA. However, epitope mapping with a panel of 24 anti-CEA MAbs (recognizing at least 10 CEA epitopes) stowed virtual immunologic identity between these two molecules. Moreover, BVCEA-140 appears to be a more potent humoral immunogen in mice than native CEA. These purified recombinant proteins can thus serve as standards in CEA serum assays for the possible detection and characterization of cell-mediated immune responses to CEA and as a potential source of immunogen (primary or for boosting) for active specific immunotherapy protocols of human carcinomas.