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Hyaluronan (HA) is a negatively charged glycosaminoglycan that exhibits a wide variety of biological effects mediated by binding to cell-surface and extracellular matrix proteins (hyaladherins). Short HA oligosaccharides have been shown to retain the specific interactions and biological effects of high molecular weight HA. Although it has a simple disaccharide repeating unit, the aqueous solution conformation of HA has been very difficult to determine because of strong coupling and overlapping resonances. In this study, we propose aqueous solution conformations for an octasaccharide of HA, derived from proton-proton NOE data and restrained molecular dynamics. To overcome spectral overlap and strong coupling, alternate methods for extracting distance restraints were employed. Restrained molecular dynamics calculations yielded one set of interglycosidic angle values for the beta (1,3) linkage (phi 13 = 46 degrees, psi 13 = 24 degrees). In contrast, two sets of values for the beta (1,4) linkage were consistent with the NOE restraints (phi 14 = 24 degrees, psi 14 = -53 degrees or phi 14 = 48 degrees, psi 14 = 8 degrees). The potential difference in flexibility for the two linkages is consistent with unrestrained as well as the restrained molecular dynamics trajectories described here. The conformational parameters obtained from restrained molecular dynamics are used to predict helical parameters of high molecular weight HA and will provide a basis for studies of HA binding to proteins.