A tumor-associated antigen (designated TEX) has been isolated from liver metastases of colon carcinoma. This antigen shares some immunological properties with the carcinoembryonic antigen (CEA), to which it appears to be related. Like CEA, TEX is a glycoprotein. It binds to concanavalin A Sepharose, from which it can be eluted by displacement with methyl α-D-mannoside. By Sephadex G-200 gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its molecular weight is shown to be 110,000 daltons. The total amount of carbohydrate in TEX was 35%, compared to 60% in CEA. Linkage analysis of the carbohydrate moieties by methylation analysis reveals that TEX contains substantially less terminal galactose, as well as less 4-linked interchain and 3,4-branched N-acetylglucosamine, when compared to CEA. The results of periodate treatment of TEX are similar to those obtained for CEA, in that all the sialic acid and fucose and 25% of the N-acetylglucosamine residues are destroyed, but the results differ in that twice as much mannose and galactose are destroyed by periodate in TEX than are destroyed in CEA. TEX is more resistant to mild acid hydrolysis than is CEA, as judged by its profile on Sephadex G-200 column chromatography and its high antigenic activity on radioimmunoassay after one cycle of a Smith degradation. By contrast CEA is degraded into peptide fragments and loses 68% of its antigenic activity. The amino acid composition of TEX is nearly identical with that of CEA, except for the presence of small but reproducible amounts of methionine in TEX, but not in CEA. The sequence of the first 24 NHrterminal amino acids of TEX shows extensive homology to CEA. The only difference identified is alanine at position 21 in TEX but valine in this position for CEA. The majority of evidence, both immunological and chemical, indicates that TEX is closely related to CEA but differs slightly in its mode and degree of glycosylation and has one or more amino acid alterations in its polypeptide chain. TEX may be identical with the normal or nonspecific cross-reacting antigen, also known as NGP, CCA-III, CCEA-2, CEX, and the βE-protein. The results reported here present data comparing the immunochemistry of CEA and of this highly purified cross-reacting antigen. © 1978, American Association for Cancer Research. All rights reserved.