The role of ongoing RNA synthesis in chromatin organization in Chinese hamster ovary cells was examined upon exposure to the transcription inhibitor alpha-amanitin. Treatment with alpha-amanitin led to pleomorphic nuclei with chromatin heavily condensed and with the remaining ribonucleoprotein aggregated in large compact granular masses around the margins of the nuclear periphery. Concommitant with the changes in nuclei morphology transient focal dilatation of the rough endoplasmic reticulum was observed while other cytoplasmic organelles appeared structurally unaffected. The morphological changes occurred after complete inhibition of RNA polymerase II mediated transcription. The molecular integrity of isolated DNA was monitored in parallel with the structural analysis. Fragmentation of cellular DNA occurred in a time-dependent fashion and well after the complete inhibition of RNA synthesis. Characteristic oligonucleosomesized DNA fragments of about 187 base pairs in length was produced in a cotemporal time-dependent fashion. Our findings indicate that ongoing transcription and the structural state of chromatin are very closely integrated, and provide further evidence that RNA is a structural component of the nuclear matrix, which in turn is involved in keeping chromatin physically dispersed and decondensed.