The murine monoclonal antibody DF3, prepared against a membrane-enriched fraction of a human breast carcinoma, recognized high molecular weight mucin-like glycoproteins from normal human milk and breast carcinoma cell lines. Although the epitope recognized appeared to be a peptide segment, recognition was altered by neuraminidase, suggesting carbohydrate contributions to antigen recognition. Examination of DF3 antigen isolated from normal human milk and the BT-20 human breast carcinoma cell line showed significant oligosaccharide differences. DF3 antigen from BT-20 cells contained three major oligosaccharides, the peanut agglutinin-binding disaccharide Gal beta 1,3GalNAc, which is the carbohydrate component of the Thomsen-Friedenreich antigen, and its mono-(NeuAc2,3Gal beta 1,3GalNAc and/or Gal beta 1,3(NeuAc alpha 2,6)GalNAc) and disialylated (NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc) derivatives. In contrast, the DF3 antigen from normal human milk contained the tetrasaccharide Gal beta 1,4GlcNASc beta 1,6(Gal beta 1,3)GalNAc as its major neutral oligosaccharide and also sialylated derivatives of the tetrasaccharide, including a monosialo derivative (Gal beta 1,4GlcNac beta 1,6(NeuAc alpha 2,3Gal beta 2,3)GalNAc and/or NeuAc alpha 2,3Gal beta 1,4GlcNAc beta 1,6 (Gal beta 1,3)GalNAc). These results suggest the possibility of carcinoma-associated alterations in O-linked oligosacchardes of cell surface sialomucins and in the activity of the beta 1,6-glucosaminyl transferase involved in mucin biosynthesis.