The　rare　N-unsubstituted　glucosamine　(GlcNH　(3)　(+)　)　residues　in　heparan　sulfate　(HS)　have　important　biological　and　pathophysiological　roles.　However,　it　is　difficult　to　prepare　naturally-occuring,　GlcNH　(3)　(+)　-containing　oligosaccharides　from　HS　because　of　their　low　abundance,　as　well　as　the　inherent　problems　in　both　excising　and　identifying　them.　Therefore,　the　ability　to　chemically　generate　a　series　of　structurally-defined　oligosaccharides　containing　GlcNH　(3)　(+)　residues　would　greatly　contribute　to　investigating　their　natural　role　in　HS.　In　this　study,　a　series　of　heparin/HS　oligosaccharides,　from　dp6　up　to　dp16　in　length　that　possess　internal　GlcNH　(3)　(+)　residues　were　prepared　by　a　combination　of　chemical　modification　and　heparinase　I　digestion.　Purification　and　structural　analysis　of　the　major　species　derived　from　the　octa-　to　dodeca-saccharide　size　fractions　indicated　the　introduction　of　between　1　and　3　internal　GlcNH　(3)　(+)　residues　per　oligosaccharide.　In　addition,　a　GlcNH　(3)　(+)　residue　was　selectively　introduced　into　an　internal　position　in　a　tetrasaccharide　species　by　direct　chemical　modification.　This　selectivity　has　potential　as　an　alternative　procedure　for　preparing　internally-modified　oligosaccharides　of　various　lengths.　The　utility　of　such　oligosaccharides　was　demonstrated　by　a　comparison　of　the　binding　of　three　different　tetrasaccharide　species　containing　0,　1　and　2　free　amino　groups　to　the　NK1　truncated　variant　of　hepatocyte　growth　factor/scatter　factor.