The monoclonal antibody (MAb) DF3 reacts with a high molecular weight glycoprotein detectable in human breast carcinomas and human milk. The present studies have analyzed the structure of DF3 protein produced by human BT-20 and MCF-7 breast carcinoma cells. The size of the DF3 core protein was determined by glycosidase digestion of purified DF3 glycoprotein and by immunoprecipitation after [3H]proline labeling. A core protein size of approximately Mr 160,000 was obtained for DF3 protein in BT-20 cells. In contrast, two DF3 proteins of approximately Mr 160,000 and 230,000 were detectable in MCF-7 cells. Amino acid analysis indicated that DF3 antigen from these cells is relatively rich in threonine, serine, proline, glycine, and alanine. The results also demonstrate that the DF3 epitope resides on the core protein, although reactivity with MAb DF3 was affected by the presence of carbohydrate. Finally, using synthetic peptides, we demonstrate that the DF3 epitope is present within the 20 amino acids encoded by the tandem repeat recently identified from our sequence analysis of a DF3 complimentary DNA.