ABSTRACT The yeast nuclear scaffold has been shown to bind with high affinity to genomic sequences that permit autonomous DNA replication of plasmids (ARS elements). We describe here conditions for the isolation of a histone-free nuclear substructure, the nuclear scaffold, from Saccharomyces cerevisiae. We examine the protein composition of this structure, and the conditions under which topoisomerase II, the nuclear factor RAP-1 and RNA polymerase II cofractionate with the scaffold. We find that exposure of nuclei to a combined metal and heat treatment (0.5 mM Cu2+, 37 °C) prior to detergent extraction is required for effective stabilization of these proteins with the scaffold. Electron microscopy of the residual nuclei extracted with non-ionic detergents shows the absence of a typical peripheral lamina structure.