Membrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F-actin: 1) small aggregates within the diffuse CNPase regions and 2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 micrograms/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F-actin is not altered under these conditions. In contrast, cytochalasin B disrupts F-actin, microtubules, and CNPase in the lacy networks, indicating that cross-linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for 1) direct association of CNPase with F-actin and tubulin in cytoskeletal structures and 2) organization of MBP into domains via association with microtubules in the lacy networks.