With the procedure which is described, brain tissue is separated into six fractions, the sum of whose weights equals the dry weight of the tissue. Three of these primary fractions are further separated into subfractions. Recovery is quantitative at each step. The procedure, as applied to rat brain, gave consistent results; duplicate analyses of the two halves of brains, divided as equally as possible along the anteroposterior line, agreed closely, and the variation from rat to rat in the percentages of the various fractions in dry tissue was small, as illustrated by the following results: main lipid fraction, 40.65 ± 1.03 (S.D.)%; protein residue, 44.45 ± 0.87%; water-soluble fraction from Folch purification, 5.94 ± 0.23%; crude "strandin" fraction, 1.25 ± 0.05%. The average percentage of dry in fresh tissue was 24.87 ± 0.41. The method includes procedures for the separation of a fraction, obtained by treatment with acidified chloroform-methanol, into four well-defined fractions, of which one is characterized by extreme surface activity in aqueous solution. A small amount of lipid is so firmly bound in brain tissue as to resist extraction during exhaustive treatment with chloroform-methanol (2:1), acidifed chloroform-methanol, and acetone. Significant radioactivity was present in most of the fractions from the brains of rats which had received a peritoneal injection of acetate-C14 5-15 months previously during the period of early development.