Negatively supercoiled plasmids can be assembled into dynamic minichromosomes using Drosophila embryo extract as a source of histones and chromatin assembly factors. However, analysis of such mini-chromosomes is often difficult due to the presence in the crude extract of a large excess of macromolecules and low molecular weight molecules including ATP. Several techniques have been used to partially purify the minichromosomes based on either sizing columns or centrifugation on sucrose gradients. We have developed a single-step method employing a 30 min ultracentrifugation through a glycerol cushion. In contrast to chromatin purified in sucrose-containing buffers, the minichromosomes obtained with this method are suitable for transcriptional analysis. This method is fast, quantitative, flexible, can deal with several samples simultaneously and leads to concentration of the chromatin. As centrifugation through glycerol yields chromatin free of ATP and several characterized chromatin remodeling complexes, this method should be useful for structural and functional studies in vitro.