The hydroxy protons of unsaturated di-, tetra-, hexa- and octa-saccharides of hyaluronan (ΔHA₂, ΔHA₄, ΔHA₆ and ΔHA₈) in 85% H₂O/15% acetone-d₆ have been studied by NMR spectroscopy. The chemical shifts (δ), chemical shift differences (Δδ), temperature coefficients (dδ/dT) and nuclear or rotating-frame Overhauser effects (NOEs or ROEs) of hydroxy protons were measured to gain insight into hydration, hydrogen bonds and flexibility of the HA structure. The NMR data give the first experimental evidence that weak hydrogen bonds exist between O(4)H of N-acetyl-D-glucosamine (GlcNAc) and O(5) of D-glucuronic acid (GlcA) across the β(1→3) glycosidic linkage and between O(3)H of GlcA and O(5) of GlcNAc across the β(1→4)-linkage. A chemical exchange was observed between O(4)H of GlcNAc and O(2)H of GlcA over the β(1→3)-linkage. The interaction could be mediated through water bridge(s) and thus contribute to the water-retaining ability of hyaluronan. In this study it was also demonstrated how the chemical shifts of exchangeable hydroxy or amide proton signals can be used to describe small structural and conformational perturbations within large oligosaccharides.