Fatty acid order parameter profiles were determined by 2H-NMR in order to characterize the arrangement and behaviour of the hydrophobic region of glycosphingolipids (GSLs) dispersed as minor components in phosphatidylcholine/cholesterol membranes. Direct comparison was made amongst species with important fatty acid structural features found in natural glycosphingolipids. Galactosyl ceramides (GalCer) were prepared by partial synthesis having 18:0[d35], D-alpha-OH 18:0[d34], 18:1[d33], and 24:0[d47] fatty acids. Unsonicated multilamellar liposomes of the common natural phospholipid, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), containing 23% cholesterol, were employed as host matrix. Smoothed profiles of the order parameter, SCD, for 18:0[d35] GalCer proved to be very similar to profiles known for 16:0 and 18:0 fatty acids of glycerolipids in cholesterol-containing bilayers. In general, order along the GSL chain was slightly higher than anticipated for equivalent chain segments in phospholipids. Order parameter profiles for the GSL 18-carbon saturated fatty acids were strikingly similar. However, small quantitative differences were found for glycolipids having D- and L-alpha-hydroxylation at C-2 - the D-stereoisomer being marginally more ordered in the plateau region. Although order profiles have not been reported for unsaturated glycerolipid fatty acids in cholesterol-rich membranes, spectra of 18:1[d33] GalCer appeared to be assignable by applying known ordering effects of cholesterol to existing data for unsaturated glycerolipids. The unsaturated chain was found to be less ordered than saturated 18-carbon chains toward the membrane surface, but more ordered in the region of the bilayer midplane. The ordering may result from cholesterol-induced restriction of isomerisation at the cis-double bond, and represents an apparent exaggeration of a phenomenon known for glycerolipids. Addition of an 'extra' 6 carbons to the fatty acid (24:0[d47] GalCer) produced no significant effect on the order profile to a membrane depth of C-12-C-13. These results suggest that fluid membrane area requirements for GSLs with saturated fatty acids are not strongly influenced by the nature of that fatty acid when the GSL is a minor component. Order parameter profiles for the very long chain GSL deviated to higher order below this point, and formed a second 'plateau' of reduced negative slope toward the methyl terminus: this is characteristic of profiles for very long chain GSLs. These features were essentially unchanged over a range of temperatures providing different degrees of spatial constraint.