Galactosyl ceramide (GalCer) was labeled for nuclear magnetic resonance (NMR) spectroscopy by replacement of a hydrogen atom at C6 of the galactose residue with deuterium. Wideline 2H NMR of [d1]GalCer permitted consideration of a mechanism traditionally entertained for cell surface recognition site modulation: that the nature of the fatty acid attached to the sphingosine backbone of glycosphingolipids (GSLs) importantly influences carbohydrate headgroup orientation. Comparison was made among various glycolipid fatty acids by altering hydroxylation, saturation, and chain length. Studies were carried out in unsonicated bilayer membranes mimicking several important characteristics of cell plasma membranes: fluidity, low GSL content, predominant [sn-2]monounsaturated phosphatidylcholine (PC) (1-palmitoyl-2-oleoyl PC), and the presence of cholesterol. Spectroscopy was performed on samples over a range of temperatures, which included the physiological. 2H NMR spectra of [d1]GalCer having 18-carbon saturated fatty acid (stearic acid), cis-9-unsaturated fatty acid (oleic acid), D- and L-stereoisomers of alpha-OH stearic acid, or 24-carbon saturated fatty acid (lignoceric acid) were importantly similar. This argues that for GSLs dispersed as minor components in fluid membranes, variation of the glycolipid fatty acid does not provide as much potential for direct conformational modulation of the carbohydrate portion as has sometimes been assumed. However, there was some evidence of motional differences among the species studied. The 2H NMR spectra that were obtained proved to be more complex than was anticipated. Their features could be approximated by assuming a combination of axially symmetric and axially asymmetric glycolipid motions. Presuming the appropriateness of such a analysis, at a magnetic field of 3.54 T (23.215 MHz), the experimental spectra suggested predominantly asymmetric motional contributions. At the higher field of 11.7 T (76.7 MHz, equivalent to a proton frequency of 500 MHz), spectra indicated dominance by axially symmetric rotational modes. There was also evidence of some bilayer orientation in the stronger magnetic field. The unusual observation of spectral differences between the two magnetic field strengths may involve a diamagnetic response to high field on the part of some liposome physical characteristics.