The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.