Using sequential digestion with the glycyl-glycine endopeptidase lysostaphin followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were purified and analyzed by a combination of reverse-phase-high pressure Liquid chromatography (HPLC) and mass spectrometry. Reverse-phase-RPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharides composed of repeating disaccharide units (N-acetylglucosamine-[beta-1,4]-N-acetylmuramic acid) with different degrees of polymerization and terminating with N-acetylmuramic acid residues at the reducing ends. The method allowed separation of strands up to 23-26 disaccharide units with a predominant length between 3 and 10 and an average degree of polymerization of similar to 6. Glycan strands with a higher degree of polymerization (>26 disaccharide units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of minor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest that the satellites are the products of an N-acetylglucosaminidase activity that differs from the atl gene product and that appears to be involved with modification of the glycan strand structure.