The structure of the pentasaccharide S259-1 in the Consortium for Functional Glycomics was investigated using a variety of techniques. Surprisingly, the structure differs from the structure assumed from the previously established specificity of the human fucosyltransferase FUT-III used in the last step of chemoenzymatic synthesis. When presented with a tetrasaccharide substrate containing both type I and type II disaccharide moieties, the enzyme generates a pentasaccharide in which the type II moiety is preferentially fucosylated. The unexpected product generated by FUT-III in this case highlights the importance of performing detailed structural analysis on products generated by enzymes.