AbstractMyelin isolated from three areas of mouse brain, from whole brain at several ages in normal mice, and from whole brain of adult quaking mutant mice was separated into seven bands and a pellet on discontinuous density gradients using 0.32, 0.45, 0.55, 0.60, 0.70, 0.75, and 0.85Msucrose. The distribution of myelin in the subfractions was independent of homogenization and shocking conditions employed to isolate the myelin preparations, but was related to the type of myelin applied to the gradient. Compared to myelin isolated from older animals, myelin isolated from 18–24 day old mice displayed a distribution pattern with greater proportions of material banding at lesser sucrose densities. Similarly, myelin obtained from hindbrain contained proportionately more material layering at lesser sucrose densities compared to myelin isolated from cerebral cortex. Myelin subfraction patterns observed for 8–12 day old control mice and quaking mutants were unlike each other or any other myelin preparation examined.In the 18–90 day old animals, the markers studied were not uniformly distributed among the myelin subfractions. The pellet and the layer banding at the 0.75/0.85Msucrose interface contained the highest specific concentrations of sialic acid, nucleic acid, and total adenosine triphosphatase activity. In contrast, the specific activity of 2′,3′ ‐cyclicnucleotide‐3′‐phosphohydrolase was lowest in the pellet as well as the three bands obtained above 0.60Msucrose and was highest in the fraction banding at the 0.65/0.70Msucrose interface.The results obtained were not consistent with an artifactual origin of the myelin subfractions, but instead suggested that the subfractions have physiological significance. One explanation for the different banding patterns observed between young and mature myelin may be the different amount of myelin in various brain regions d