One level of DNA organization in metaphase chromosomes is brought about by a scaffolding structure that is stabilized by metalloprotein interactions. Fast-sedimenting, histone-depleted structures (4000-7000 S), derived from metaphase chromosomes by extraction of the histones, are dissociated by metal chelators or by thiol reagents. The chromosomal (scaffolding) proteins responsible for constraining the DNA in this fast-sedimenting form are solubilized under the same conditions. Chromosomes isolated in a metal-depleted form, which generate slow-sedimenting, histone-depleted structures, can be specifically and reversibly stabilized by Cu2+, but not by Mn2+, Co2+, Zn2+ or Hg2+. Metal-depleted chromosomes can also be stabilized by Ca2+ (at 37 degrees C), but this effect is less specific than that of Cu2+. The scaffolding protein pattern that is reproducibly generated following treatment with Cu2+ is composed primarily of two high molecular weight proteins--Sc1 and Sc2 (170,000 and 135,000 daltons). The identification of this simple protein pattern has depended upon the development of new chromosome isolation methods that are highly effective in eliminating cytoskeletal contamination.