Myelinating Schwann cells polarize their surface membrane into several ultrastructurally and biochemically distinct domains that constitute the myelin internode. Formation of these membrane domains depends on contact with appropriate axons and requires microtubule-based transport systems for site-specific targeting of membrane components. Because little is known about microtubules in myelinating Schwann cells, this study used confocal microscopy and the microtubule hook-labelling method to characterize microtubule distribution, the location of microtubule nucleation sites, and the polarity and composition of Schwann cell microtubules. In myelinating Schwann cells, microtubules were abundant within the Golgi-rich perinuclear cytoplasm; they were not attached to the centrosome. Three-fourths of the microtubules in the cytoplasmic channels located along the outer perimeter of the myelin internode had their (+) ends oriented away from the perinuclear region, whereas the remaining 25% had the opposite polarity. Depolymerization/repolymerization experiments detected microtubule nucleating sites in perinuclear cytoplasm but not along the myelin internode. Taken together, these results indicate that microtubule-mediated transport of myelin components along the internode could utilize both (+)- and (-)-end motors. Specialized microtubule tracks that target myelin proteins to specific sites were not identified on the basis of tubulin polarity or posttranslational modifications.