The organization of epithelial cells in distinct areas of the thymus appears important for understanding the pathways of T-cell differentiation. The presence of carcinoembryonic antigen (CEA) was investigated on sections of human thymus and in cultures of thymic epithelial cells through use of monoclonal antibodies (Mab) discriminating CEA and 2 non cross-reacting antigens (NCA 55 and NCA 95). All together, 5 monoclonal antibodies were used. The Mab 35 and 73 recognized exclusively a specific CEA antigenic determinant, whereas Mab 47, 192 and 202 were also reactive with CEA cross-reacting antigens. By immunofluorescence or the immunoperoxidase technique, staining of CEA was restricted essentially to Hassall's corpuscles and a few adjacent epithelial cells on thymic sections; this pattern was similar to distribution of Ca 19-9 antigen. Additionally, the 47, 192 and 202 antibodies were reactive with a keratin-negative subset mainly located in the cortex. Furthermore, a subset of keratin-positive cells bearing CEA molecules was observed in thymic epithelial cell cultures. Positive cells comprised less than 3% with Mab 35 and 73, and reached up to 12% with Mab 47, 192 and 202. By flow cytometry analysis, staining intensity varied with the epitope; it was much weaker with Mab 35, 47 and 73 than with Mab 192 and 202. The CEA content in culture supernatants was inversely correlated to the number of CEA positive cells. Thus, CEA could be considered as a marker of a late maturation stage of medullary epithelial cells culminating in Hassall's corpuscles and could contribute to delineating the heterogeneity of thymic epithelial cells.