Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergentresistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-␤-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-␤-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.Recent studies suggest that plasma membrane lipids do not always mix homogeneously but that membranes may contain microdomains or rafts that are rich in sphingolipids and cholesterol (1-3). Rafts may be concentrated or stabilized in caveolae but also exist in cells that lack caveolae. Rafts have been proposed play important roles in signal transduction; for instance, recruitment of signaling proteins to rafts in T cells (4 -8) and basophils (9 -11) appears to be required for signaling. Rafts may also play a role in intracellular sorting. For instance, depletion of cholesterol, sphingolipids, or certain raftassociated proteins affects sorting of apical proteins in epithelial cells (12-17) and axonal proteins in neurons (18,19). Recent studies also suggest that rafts play a role in sorting in the endocytic pathway (20,21).Two models for the organization of lipids in rafts have been proposed. The first was developed by Simons and colleagues (1,22,23) as part of a model for sorting of apical and basolateral proteins in the trans-Golgi network of polarized epithelial cells. In this model, clusters of GSL 1 form spontaneously in the trans-Golgi net...