A method for isolating pure and unaltered nuclei from rat brain by means of differential centrifugation is described. The isolated nuclei are further separated into discrete fractions of neuronal, astrocytic, and glial nuclei, with a yield amounting to 20 to 25% of the DNA of the original homogenate. Both the morphology and size of the nuclei remained unchanged. Problems concerning the composition of the isolation media, the use of detergents, as well as those raised by density gradient centrifugation in sucrose, Ficoll, and Dextran are discussed. Some values for the density of each type of brain nuclei are suggested.