Abstract— A procedure is described for the preparation from rat brain of myelin having the same degree of purity at all ages. Such a procedure is essential for the study of myelin composition during development. Microsomal contamination was successfully eliminated by adjusting the method to maintain a constant amount of brain per unit volume in the initial density gradient step, and by including two osmotic shocks and two low-speed centrifugation steps. Myelin prepared in this way from animals ranging from 15 days to 14months of age had a total ATPase activity of 0.3-2.0 μmol of Pi.h−1.mg−1 dry wt of myelin, representing 0.1-1.2 per cent recovery of the total homogenate activity; a Na+, K+- ATPase activity of 0.1-1.6 μfnol of Pi.h−1.mg−1 dry wt, representing 0.04-1.5 per cent recovery; a nucleic acid content of 0.2-0.7 per cent of myelin dry wt, representing 0.2-2.0 per cent recovery; and a ganglioside NANA content of 0.04-0.07 per cent of myelin dry wt. representing 0.2-4.6 per cent recovery. The myelin prepared from 20-day animals had the highest content of the first three constituents; otherwise the values of the four constituents were relatively constant per unit weight of myelin. The amounts of nucleic acid and ganglioside recovered in the myelin fractions increased with increasing age and myelin yield.