Six mouse monoclonal antibodies reactive with carcinoembryonic antigen (CEA) were prepared and used for the analysis of the antigenic heterogeneity of CEA in patient sera. Their reaction specificity and the chemical nature of antigenic epitopes recognized by them were analyzed by radioimmunoassay on the basis of reactivities with different preparations of CEA, normal fecal antigen 2, and nonspecific cross-reacting antigen 2 before and after chemical and/or enzymatic treatment. Two antibodies, F3-30 and F4-82, raised with CEA were reactive with different peptide epitopes on the antigen molecules and revealed a quite universal reactivity with all CEA, normal fecal antigen 2, or nonspecific cross-reacting antigen 2 preparations tested. The serum CEA values obtained with these antibodies were highly correlated with those obtained with conventional radioimmunoassays for CEA. The other four antibodies (F4-11 and F33-37 raised with CEA, F8-52 with normal fecal antigen 2, and F48-60 with nonspecific cross-reacting antigen 2) were found to recognize carbohydrate epitopes with different specificities and revealed very heterogeneous reactivities. The serum CEA values estimated with these four antibodies were highly variable depending on the antibody used, suggesting that the expression of carbohydrate epitopes on the CEA molecules in patient sera was quite heterogeneous. The antigenic heterogeneity of the carbohydrate epitopes was detected even in a single patient serum by affinity chromatography. The causes that give rise to the difference in CEA values between the Roche and the Daiichi kits were analyzed on the basis of reactivities of three groups of patient sera, which showed extremely different ratios for the Roche and Daiichi kits, with monoclonal anti-carbohydrate antibodies. The results obtained suggest that, at least in part, the diversity of antigenic expression on carbohydrate chains on the CEA molecules in patient sera and the variation in specificity or quantity of anti-carbohydrate antibodies in the polyclonal antibody preparations used for the respective assay systems may result in the differences in the estimated CEA values.