The ¹³C resonances of the glycerol and choline groups of dipalmitoyl-lecithin in CD₃OD, CDCl₃, and D₂O have been assigned from the ¹⁴N–¹³C and ³¹P–¹³C coupling constants, and proton decoupling studies on the assigned ¹H spectra. Well-defined solvent effects are observed, but the chemical shifts for dipalmitoyl-lecithin in bilayers in D₂O are very similar to the shifts of the corresponding resonances in biological membranes. From an analysis of the ¹H spectrum of the CH₂ multiplet in the CH–CH₂OCOR fragment, the two vicinal coupling constants were extracted and used to calculate rotamer populations from a Karplus treatment. In a high proportion of the molecules of dipalmitoyl-lecithin in CD₃OD and in CDCl₃ the fatty acid chains are arranged gauche to each other, suggesting the presence of strong hydrophobic interactions. From an AA′BB′ analysis of the choline –CH₂·CH₂–fragment in dipalmitoyl-lecithin observed vicinal coupling constants indicate a similar conformation to that found in choline derivatives where an electrostatic interaction between the N⁺ and the O atoms leads to the conformation being exclusively in the gauche⁺N/O form.