Factors which influence the accessibility, or exposure, of the carbohydrate head group of the glycolipid galactosylceramide (GalCer) at the membrane surface have been examined in lipid model membranes using the technique of galactose oxidase-tritiated sodium borohydride labeling. Both the ceramide composition of GalCer and the lipid composition of its membrane environment were varied. We have shown that GalCer is oxidized in a membrane environment, by purification of the labeled galactosyl moiety of the glycolipid by high-performance anion exchange chromatography. Using semisynthetic molecular species of GalCer with acyl chain lengths ranging from 16 to 26 carbons, incorporated into liposome membranes of egg phosphatidylcholine (PC), and reverse-phase HPLC separation of mixtures of the molecular species, we have shown that increasing the fatty acid chain length of GalCer increases its oxidation by galactose oxidase. In addition, the degree of oxidation is reduced when the fatty acid chain of GalCer is hydroxylated. GalCer incorporated into liposomes containing synthetic species of PC with different fatty acid chain lengths (together with cholesterol) was oxidized less as the PC acyl chain length, and hence the bilayer thickness, was increased. The oxidation of GalCer in liposomes composed of sphingomyelin/cholesterol was reduced compared to its oxidation in PC liposomes. Furthermore, changes in the fatty acid chain length of GalCer had no effect on its oxidation in sphingomyelin liposomes. These findings indicate that the ceramide composition and lipid membrane environment can influence the exposure of the lipid carbohydrate, and hence, they could modulate the receptor activity of glycolipids at the membrane surface.