We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gel patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.