The Thomsen‐Friedenreich (T) antigen and its disaccharide component Galβ1,3GalNAc, which is recognized by the plant lectin peanut agglutinin (PNA), have been proposed as useful tumor markers because of their apparently specific occurrence in certain types of carcinomas. We have investigated the mechanism for the appearance of the disaccharide at the cell surface of ascites 13762 rat mammary adenocarcinoma cells using pulse‐chase glucosamine labeling, proteolysis, and PNA precipitation of the cell‐surface sialomucin ASGP‐1. Glucosamine‐labeled disaccharide appears at the cell surface in less than 10 min. Although the appearance of larger oligosaccharides continues to increase, the appearance of labeled disaccharide levels off within an hour. Analysis of intracellular vs. cell surface‐labeled oligosaccharides showed that all disaccharide synthesized more than an hour before reaching the cell surface is converted to larger oligosaccharides. Thus, the presence of the disaccharide at the cell surface results from its synthesis late in the transit pathway of the sialomucin tb the cell surface. We propose that the presence of T antigen at the surface of carcinoma cells results from an aberration of the pathway for O‐linked glycosyiation in these cells, probably caused by inappropriate localization of the enzymes involved in synthesis of the disaccharide.—Hull, S. R.; Carraway, K. L. Mechanism of expression of Thomsen‐Friedenreich (T) antigen at the cell surface of a mammary adenocarcinoma.FASEB J.2: 2380‐2384; 1988.