The conformational preference of the human milk oligosaccharide lacto-N-neotetraose, beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-(1 -> 3)-beta-D-Galp-(1 -> 4)-D-Glcp, has been analyzed using H-1,H-1 T-ROESY and H-1,C-13 trans-glycosidic J coupling experiments in isotropic solution and H-1,13C residual dipolar couplings (RDCs) obtained in lyotropic liquid crystalline media. Molecular dynamics simulations of the tetrasaccharide with explicit water as the solvent revealed that two conformational states are significantly populated at the psi glycosidic torsion angle, defined by C-anomeric-O-C-H, of the (1 -> 3)-linkage. Calculation of order parameters, related to the molecular shape, were based on the inertia tensor and fitting of experimental RDCs to different conformational states showed that psi(+) > 0 degrees is the major and psi(-) < 0 degrees is the minor conformation in solution, in complete agreement with a two-state analysis based on the T-ROESY data. Attention was also given to the effect of salt (200 mM NaCl) in the anisotropic medium, which was, a ternary mixture of n-octyl-penta(ethylene glycol), n-octanol, and D2O.