Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after heparin lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies.