The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane. Three protocols are outlined in this unit: isolation of a highly‐purified myelin fraction from the central nervous system (CNS); separation of a highly‐purified CNS myelin fraction into subfractions of different densities; and isolation of myelin from the peripheral nervous system (PNS).