The chemical structure of carcinoembryonic antigen (CEA) and two closely related antigens, normal fecal antigen-2 (NFA-2) in normal adult feces and nonspecific cross-reacting antigen-2 (NCA-2) in the meconium, were further analyzed comparatively. The NH2-terminal amino acid sequence of NCA-2 was newly determined to position 18 and found to be identical to that so far determined for CEA- and NFA-2. After proteolytic digestion with chymotrypsin or protease V8, the digests of these antigens showed two groups of fragments upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One consisted of the sharply banded fragments which were identical in all antigens and stained only with Coomassie brilliant blue (CBB) (five bands in the range 2500-10,000 daltons for chymotrypsin and 11 bands in the range 8000-35,000 daltons for protease V8, respectively), and the other consisted of the dispersed fragments which had variable mol. wts in the range 10,000-100,000 and were stainable with both CBB and periodic acid-Schiff reagent. Elution profiles of CEA, NFA-2, and NCA-2 from lectin columns, especially from concanavalin A-Sepharose columns, suggested some differences in oligosaccharide chains between them. These results indicate that the fundamental chemical structure of these antigens seems to be very similar to one another and is divided into two parts; an homologous portion(s) which is common to all three antigens and contains no sialylated sugar components, and a heterogeneous portion(s) which is variable among these antigens and contains sialylated sugar components.