Publisher Summary The use of cell-free experimental systems in which the topology of cellular organelles is preserved provides a powerful tool in the study of cellular processes. This chapter describes the mammalian nuclear reassembly system that utilizes tissue culture cells synchronized in prometaphase. In the simplest procedure, nuclear envelope reassembly occurs about endogenous chromosomes and chromosome clumps present in a homogenate of these synchronized cells. However, assembly will also take place around exogenous purified chromosomes in which a postchromosomal supernatant of the mitotic homogenate provides all necessary nuclear envelope components. The initiation of the nuclear envelope assembly process in mammalian mitotic cell homogenates is found to be strongly inhibited by adenosine triphosphate (ATP) and ATP-γ-S. This indicates that the dephosphorylation of a key regulatory molecule, for example, cyclin-dependent kinase, is required to get the process started. ATP levels do not vary appreciably during the cell cycle; the initiation of nuclear envelope assembly in the mitotic homogenate is likely to represent a nonphysiological occurrence. However, the subsequent steps in the assembly process appear to closely parallel events observed in vivo and certainly give rise to ultrastructurally normal nuclear envelopes surrounding decondensed chromatin.