The solution conformation of homogeneous, heparin-derived hexasaccharide (residues A, B, C, D, E, F) has been investigated by using 1H-NMR spectroscopy. Intra-ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various intraring forms. All glucosamine residues B, D and F are in the 4C1 chair conformation. The uronate (A) residue is mostly represented by the 1H2, form, whereas internal iduronates (C and E) exist in equilibrium between the chair and skewed boat forms. Deviations in some NOEs indicate a minor contribution of the 2H1, form to the A ring. Glycosidic dihedral angles, which define the overall oligosaccharide conformation, were further refined by combining in vacuo energy map calculations and restrained energy minimization in explicit solvent water. Conformational stability was further assessed by subjecting NOE and IRMA-derived structures to 600 ps of unrestrained molecular dynamics in explicit solvent.