The high degree of structural order inside the nucleus suggests the existence of an internal nucleoskeleton. Our studies on the east gene of Drosophila, using the larval salivary gland polytene nucleus as a model, demonstrate the involvement of an extrachromosomal nuclear structure in modulating nuclear architecture. EAST, a novel ubiquitous protein, the product of the east (enhanced adult sensory threshold) locus, is localized to an extrachromosomal domain of the nucleus. Nuclear levels of EAST are increased in response to heat shock. Increase in nuclear EAST, whether caused by heat shock or by transgenic overexpression, results in the expansion of the extrachromosomal domain labelled by EAST, with a concomitant increase in the spacing between chromosomes. Moreover, EAST functions to promote the preferential accumulation of the proteins CP60 and actin in extrachromosomal regions of the nucleus. We propose that EAST mediates the assembly of an expandable nuclear endoskeleton which, through alterations of its volume, can modulate the spatial arrangement of chromosomes.