Publisher Summary This chapter describes a method for the isolation of nuclei from animal cells in culture. The objective of the method for isolating nuclei is to obtain a preparation that consists solely of nuclear material and in which the nuclei are morphologically identical to those in undisrupted cells, with contents qualitatively and quantitatively identical to those of nuclei in vivo. The chapter discusses the factors of importance in nuclear isolation procedures and the criteria that used to assess the integrity and purity of the isolated nuclei. The chapter describes two methods for isolating nuclei as preliminary steps in the isolation of chromatin and chromatin constituents. Nuclear isolation procedures essentially consist of two steps: (1) the lysis of the cells in a suitable medium, and (2) the separation of the nuclei from the unbroken cells and cell debris. Examination of the isolated nuclei by high-resolution electron microscopy is the best method of assessing the integrity of the nuclei and the purity of the preparation. The methods for isolating nuclei include isolation with (1) citric acid, and (2) sucrose and detergent. The chapter also compares the products obtained from these methods.