Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the mammalian central nervous system, is located on the surface of the oligodendrocyte plasma membrane and the outermost lamellae of mature myelin; it is expressed during the latter steps of myelinogenesis. It has been shown that MOG may play a pathological role in autoimmune demyelinating diseases of the central nervous system, although its physiological function remains unknown. MOG is an integral membrane glycoprotein with an extracellular immunoglobulin‐like domain and two hydrophobic segments which were predicted to be membrane‐spanning on the basis of hydropathy analysis. As a first step in elucidation of MOG function, we have investigated its membrane topology, combining immunofluorescence studies on cultured oligodendrocytes and MOG‐transfected Chinese hamster ovary cells with biochemical analyses, including in vitro translation, membrane insertion and protease‐digestion assays. Our results indicate that the C‐terminal tail of MOG is located into the cytoplasm, and that only the first hydrophobic region of MOG spans the membrane whereas the second hydrophobic region appears to be semi‐embedded in the lipid bilayer, lying partially buried in the membrane with its N‐terminal and C‐terminal boundaries facing the cytoplasm.