A cell line derived from the mouse colon adenocarcinoma, MC-38, has been transduced with a retroviral construct containing complementary DNA encoding the human carcinoembryonic antigen (CEA) gene. MC-38, which forms tumors in syngeneic C57BL/6 mice, has been extensively studied as a target for active immunotherapy. Individual transduced clones that express high levels of cell surface CEA were isolated, and two clones, termed MC-38-ceal and MC-38-cea2, were extensively characterized. The levels of CEA found on the surface of these clones were considerably higher than that found in a moderately differentiated human colon carcinoma cell line (WiDr) and were comparable to those found on the human colon carcinoma cell lines GEO and CBS (among the highest CEA-expressing cells reported). Further analysis demonstrated that the CEA expressed in the MC-38-cea1 clone had a similar molecular weight to native CEA (Mr 180,000), but the MC-38-cea2 cell line expressed a single Mr 70,000 glycosylated immunoreactive product. Seven anti-CEA monoclonal antibodies were found to react with both clones. The CEA gene present in the MC-38-cea2 clone was partially sequenced and was found to contain a deletion of two of the three repeated domains present in CEA. These results provide a basis for future studies to map immunodominant epitopes of CEA and to develop a syngeneic model system that may aid in the design of reagents and protocols to study active and passive immunotherapy directed against a carcinoma expressing human CEA.