We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used [1H,1H] NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear [1H,13C] NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on 13C chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha (2----6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the alpha (2----6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with phi angles at -60 degrees or 180 degrees and of the 3 staggered rotamers of the omega angle with a predominant gt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.