Nine monoclonal antibodies reacting with carcinoembryonic antigen (CEA) were produced after immunization of mice with either purified CEA or a CEA-producing human cell line. Their specificities were assessed by immunohistochemistry on tissue sections of neoplastic and nonneoplastic lesions. These monoclonal antibodies have different patterns of tissue reactivity. Two of them, D14 and B18, were found to have a high degree of specificity for colonic carcinoma and did not react with formalin-fixed paraffin-embedded sections of normal colon with standardized staining conditions. Most cases of noncolonic adenocarcinomas and normal epithelial structures were not stained by these two monoclonal antibodies. The specificity of the monoclonal antibodies was further investigated immunochemically using intact, reduced, and alkylated or chemically fragmented CEA. Liquid phase radioimmunoassays and antibody competition immunoenzymatic assays confirmed that the antibodies recognize different epitopes of CEA. These data support the concept of CEA heterogeneity and the reactivity of the D14 and B18 monoclonal antibodies with colonic adenocarcinomas indicates that they are useful immunohistochemical probes.