The high resolution 1H and 13C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and α-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D 2O. From the coupling constants of the sugar ring protons, a 4C 1 conformation can be deduced. In contrast to the conformation in aqueous solution, the C 6 hydroxymethylene group is freely rotating around the C 6C 5 bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C 1′ to the C 2′ methine proton of the sphingosine indicates a restricted rotation around the C 1′C 2′ bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.