Two forms of histone-depleted HeLa nuclei have been characterized (Lebkowski & Laemmli, 1982). In the studies presented here, the proteins associated with both structures are identified. Type I structures, which are obtained by extraction of isolated nuclei with a buffer containing 2 m-NaCl or dextran sulfate/heparin, retain 10 to 15% of the total nuclear proteins. These proteins are represented by the three nuclear lamina proteins of 60,000 to 70,000 M_r and numerous high molecular weight species. Histone-extraction in the presence of β-mercaptoethanol leads to more expanded type II structures. Accompanying this further DNA unfolding, is a selective loss of certain proteins that are associated with type I structures. Present in type II structures are 3 to 5% of the total nuclear proteins, almost exclusively represented by the three nuclear lamina proteins and two minor proteins of 64,000 and 200,000 M_r. The proteins of both type I and type II histone-depleted nuclei are comparable to those that remain as residual nuclear structures after histone-depletion of nuclease-digested nuclei. These residual protein structures are termed type I and type II nuclear scaffolds. The selective removal of proteins by β-mercaptoethanol is pH-dependent and maximal at alkaline pH values. It is further demonstrated that type I structures are stabilized by CaCl₂. Nuclei incubated with CaCl₂ are resistant to extraction with β-mercaptoethanol. In addition, evidence is presented that confirms that β-mercaptoethanol acts to disrupt metalloprotein interactions important for the stabilization of type I structures. The metal chelator, 1,10-phenanthroline, causes solubilization of the same set of proteins as β-mercaptoethanol. DNA binding studies are presented which show that 12 proteins of type I and four proteins of type II scaffolds bind DNA in vitro. The three nuclear lamina proteins are the major DNA binding proteins of both type I and type II scaffolds.