AbstractOligodendrocytes (OLs) and the myelin produced by them are enriched in two glycosphingolipids, galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS). We showed earlier that these two glycolipids in opposed liposomal membranes or in methanol solution can adhere to each other. Here we have examined the potential effect of an interaction between GalC/CBS in apposed membranes of oligodendrocytes (OLs) by incubating cultured OLs with GalC/CBS‐containing liposomes and observing the effect on the membrane sheets produced by OLs and on the distribution of OL constituents using fluorescent antibodies and confocal microscopy. The GalC/CBS‐containing liposomes caused redistribution or a decrease in the density of anti‐GalC and anti‐MBP staining but had no effect on the density or distribution of staining by anti‐PI(4,5)P2 that remained uniformly distributed in the membrane sheets. There was no apparent change in the area of the membrane sheets nor in the amount of MBP in OL membranes, as determined by slot blots. In addition, the GalC/CBS‐containing liposomes caused depolymerization of microtubules and actin filaments suggesting that the interaction of GSL‐containing liposomes with the extracellular surface of the OL caused transmission of a signal across the membrane. Because these two glycolipids can adhere to each other across apposed membranes, the liposomal glycolipids may be interacting with a GalC/CBS‐enriched signaling domain in the OL plasma membrane. J. Neurosci. Res. 66:242–253, 2001. © 2001 Wiley‐Liss, Inc.