Enriched cultures of rat brain oligodendrocytes were extracted with a buffer that separated the cells into a Triton X-100-soluble fraction and an insoluble cytoskeleton (CSK) residue. The buffer was optimised so that intact microtubules were preserved in the CSK residue. The partition of four myelin proteins between the soluble and the CSK fractions was determined by immunoblotting and immunofluorescence. Immunoblotting showed that two integral membrane proteins of myelin, the proteolipid protein (PLP) and the DM-20 protein, were completely extracted under these conditions. By contrast, a substantial amount of myelin basic protein (MBP) and to a lesser extent 2,3-cyclic nucleotide-3-phosphohydrolase (CNP) remained associated with the CSK residue. The association of these proteins with the CSK was confirmed by immunofluorescence. A remarkable difference in the distribution of microfilaments and microtubules was observed in oligodendrocytes. Immature cells possessed many fine processes that were rich in microfilaments. The cell body of these oligodendrocytes was devoid of microfilaments but did contain microtubules. Furthermore, a close association between CNP and microfilaments and between MBP and microtubules was revealed after detergent lysis. The strong interaction between CNP and filamentous actin was underlined by their concomitant disappearance from the extremities of the cell at a later stage of development when extensive membrane sheets had formed. Mature cells had fewer, thicker processes than younger cells and their processes contained microtubules, not microfilaments. MBP was present throughout the thick processes and the membrane sheets. These observations suggest roles for CNP and MBP at distinct stages of myelin process formation and support a directive role for the oligodendrocyte's CSK in the formation of myelin.