The myelin-associated glycoprotein (MAG) is a member of the immunoglobulin gene superfamily that is selectively expressed by myelin-forming cells. A developmentally regulated, alternative splicing of a single MAG transcript produces two MAG polypeptides (72 and 67 kD) in the central nervous system (CNS). MAG occurs predominantly as the 67-kD polypeptide in the peripheral nervous system (PNS). This study determined the subcellular localization of CNS MAG at different postnatal times when the 72-kD form (7-d) and 67-kD form (adult) are quantitatively abundant. These distributions were also compared to those of MAG in the PNS. In adult rat, MAG is selectively enriched in periaxonal membranes of CNS myelin internodes. This restricted distribution differs from that in PNS myelin internodes where MAG is also enriched in paranodal loops, Schmidt-Lanterman incisures, and mesaxon membranes. In 7-d-old rat CNS, MAG was associated with periaxonal membranes during axonal ensheathment and enriched in Golgi membranes and cytoplasmic organelles having the appearance of multivesicular bodies (MVBs). MAG-enriched MVBs were found in oligodendrocyte perinuclear regions, in processes extending to myelin internodes, and along the myelin internode in outer tongue processes and paranodal loops. MAG-enriched MVBs were not found in oligodendrocytes from adult animals or in myelinating Schwann cells. These findings raise the possibility that the 72-kD MAG polypeptide is associated with receptor-mediated endocytosis of components from the periaxonal space or axolemma during active stages of myelination.