The chemical structures of the two modified folates present in Thermococcus litoralis were established. These compounds, each containing a core structure of 1-[4-[[1-(2-amino-7-methyl-4-oxo-6-pteridinyl)-ethyl]ami-no]phenyl]-1-deoxy-[1-alpha-D-ribofuranosyl]-ribitol, were characterized. The five position of the ribose in this core structure was beta-linked to the C-1 of a poly-beta(1-->4)N-acetylglucosamine having a chain length of four or five N-acetylglucosamine residues. Thus, these compounds are N-acetylglucosamine homologs of the modified folates found in Pyrococcus furiosus.