The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have exmned the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large dumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-,-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.The eukaryotic nucleus remarkably packages more than a yard of DNA into a 5-,um spheroid. The packaging of DNA into chromatin and of chromatin into the nucleus is highly ordered (1-6), with the coarse features of this organization correlating with transcriptional activity. The condensed chromatin (heterochromatin) is largely inactive, and transcription is localized in the extended, dispersed euchromatin. The basic packing structures, the nucleosomes, are arranged in polynucleosome chains, which are wound into 30-nm fibers (7). Much less is known about the packing of chromatin fibers into higher order structures in the nuclear interior.There is a clear dependence of chromatin architecture on ongoing RNA metabolism; inhibition of RNA synthesis results in the retraction of chromatin from the nuclear lamina and its aggregation into massive clumps (8), whereas the spatial distribution of stained DNA in high-salt-extracted nuclei is changed by treatment with RNase (9). In what may be a related phenomenon, RNA synthesis inhibitors cause a retraction of polytene chromosome puffs (10,11).Many studies have described an internal structural framework in the nucleus called the nuclear matrix (12)(13)(14)(15)(16)(17), which is associated with many important chromatin functions, including DNA replication (18, 19), RNA synthesis and p...