The fine structure of acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica , was examined. This glycosaminoglycan has a major disaccharide repeating unit of -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1--> (where GlcNpAc is N -acetylglucosamine, IdoAp is iduronic acid, and S is sulfate) making it structurally related to both heparin and heparan sulfate. Using heparin lyases prepared from Flavobacterium heparinum and a newly isolated heparinase from Bacteroides stercoris , the controlled enzymatic depolymerization of acharan sulfate was undertaken to prepare a mixture of oligosaccharides. Fractionation of this mixture of oligosaccharides by strong-anion-exchange high performance liquid chromatography afforded oligosaccharides that capillary electrophoresis established were sufficiently pure for structural characterization. Electrospray ionization mass spectrometry identified two series of oligosaccharides, one derived from acharan sulfate's major repeating unit and a second minor group of undersulfated oligosaccharides. Proton nuclear magnetic resonance spectroscopy established the structure of these two classes of oligosaccharides to be DeltaUAp2S(1-->[4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S (1-->]n4)- D-GlcNpAcalpha,beta (where n = 0,1,2,3 and DeltaUAp is 4-deoxy-alpha-L- threo -hex-4-enopyranosyluronic acid) and DeltaUAp(1-->[4)- alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1-->]m-D-GlcNpAcal pha,beta (where m = 1,2,3). These results suggest the presence of minor sequence variants in acharan sulfate containing unsulfated iduronic acid having the structure -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp(1-->.