The preferred conformations and conformational flexibilities of the trisaccharides alpha-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe (I) and alpha-D-Glcp-(1-->3)[beta-D-Glcp-(1-->4)]-alpha-D-Glcp-OMe (II) in aqueous solution were determined using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) and Langevin dynamics (LD) simulations, and hydrodynamics calculations. Both trisaccharides have a vicinal substitution pattern in which long range (nonsequential) interactions may play an important role. LD simulation at 600 K indicated that the all-syn conformation predominated, though other conformations were apparent. NOE data and MD and LD simulations at 298 K all indicated that trisaccharide I is a single all-syn conformer in solution. Given that previous studies showed evidence of anti-conformers in beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe, this result provides an example of how changing the anomeric configuration of one residue from beta to alpha can make an oligosaccharide more rigid. Discrepancies in inter-ring distances obtained by experiment and by simulation of the all-syn conformer suggest the presence of an anti-psi conformation at the beta-(1-->4)-linkage for II. A combined analysis of measured and calculated translational diffusion constants and (13)C T(1) relaxation times yield order parameters of 0.9 for each trisaccharide. This implies that any interconversion among conformations is significantly slower than tumbling. Anisotropies of approximately 1.6 and 1.3 calculated for I and II, respectively, are consistent with the observed relatively flat T(1) profiles because the tumbling is not in the motional narrowing regime.