Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: [carbohydrate structure--see text] where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.