A single homogeneous isomeric species of carcinoembryonic antigen was isolated by reference to solubility in 0.9 M perchloric acid, isoelectric focusing, molecular exclusion chromatography, ion exchange chromatography, passage through immuno-absorbants, and isopyknic density gradient ultracentrifugation. The final product, representing approximately 1.8% of the perchloric acid soluble glycoprotein of the tumor, is homogeneous and devoid of other proteins by polyacrylamide gel electrophoresis. This single species of carcinoembryonic antigen, CEA-S, has a sedimentation velocity of 6.6, a diffusion constant of 3.05 times 10-minus 7 cm-2/sec, a mean Stokes radius of 65 A, a density of 1.41 ml/g in cesium chloride and an estimated molecular weight of 181,000, and it is devoid of detectable A or B blood-group antigens. Immunochemical studies demonstrate qualitative similarities between CEA-S and conventional carcinoembryonic antigens; however, competitive inhibition analyses demonstrate significant quantitative immunochemical differences between CEA-S and preparations of carcinoembryonic antigen. These results are consistent with the concept that CEA-S is an immunochemical isomer of carcinoembryonic antigen.