Nuclear envelopes have previously been shown to assemble spontaneously around endogenous chromosomes in cell-free homogenates of mitotic Chinese hamster ovary cells. In order to further analyze the mechanisms underlying nuclear envelope reformation and the functions of the individual nuclear lamin polypeptides, a fractionated cell-free nuclear envelope reassembly system involving purified chromosomes and either a postchromosomal supernatant or a cytosol fraction from mitotic cells has been devised. Results obtained with this fractionated system show that lamins A and C will associate with the surfaces of chromosomes in the absence of lamin B and membranes, this association being inhibitable by ATP-gamma-S. However, in the absence of membranes chromatin decondensation never occurs. Using the reversible swelling of chromosomes in low ionic strength buffers lacking divalent cations as the basis of a simple assay, it is demonstrated that the association of lamins A and C with the surfaces of chromosomes has a pronounced and easily observable effect on chromatin organization.