The conformation and molecular packing of permethylated /3-D-galactosyl-N-octadecanoyl-D-sphingosine (cerebroside) was determined by X-ray single crystal analysis at 185 K (R = 0.16). The lipid crystallizes in the orthorhombic space group P2~2~2~ with the unit cell dimensions a = 8.03, b ---7.04 and c = 88.10 /~. The four molecules in the unit cell pack in a bilayer arrangement with tilting (48 Β°) hydrocarbon chains. The direction of the chain tilt alternates in the two bilayer halves and in adjacent bilayers. In order to define the effect of hydrogen bonds on the molecular conformation the structural features of the permethylated cerebroside are compared with that of unsubstituted cerebroside (I. Pascher and S. Sundell (1977) Chem. Phys. Lipids 20, 179). It is shown that methylation of the hydrogen donor groups does not affect the conformation of the ceramide part. However, by abolishing the intramolecular hydrogen bond between the amide N--H group and the glycosidic oxygen the galactose ring changes its orientation from layer-parallel to layer-perpendicular. Calculations using molecular mechanics, MM2(87), show that in natural cerebroside the intramolecular hydrogen bond stabilizes the 0~ = -syn-clina/ conformation about the C(1)--C(2) sphingosine bond by 2--2.5 kcal/mol compared to other staggered conformations. The significance of the L shape of the native cerebroside, making both the carbohydrate and polar ceramide groups accessible as a binding epitope in recognition processes, is discussed.