Cockayne syndrome (CS) is a human hereditary disorder characterized by UV sensitivity, developmental abnormalities and premature aging. CS cells display a selective deficiency in transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) that preferentially removes lesions from transcribed strands. Following UV irradiation, the recovery of RNA synthesis is abnormally delayed in CS cells in conjunction with TCR deficiency. To date, TCR has been detected in cultured cells, but not in cell-free systems. In this study, we constructed an assay system using isolated nuclei. RNA synthesis catalyzed by RNA polymerases (pol I and II) was measured in nuclei prepared from UV-irradiated cells. In nuclei isolated from HeLa and xeroderma pigmentosum (XP) group C cells, RNA synthesis was relatively resistant to UV irradiation. In contrast, RNA synthesis by pol I and, in particular, pol II in CS-B nuclei was significantly inhibited upon UV irradiation. Our data support the utility of this assay system for the in vitro detection of the recovery of RNA synthesis in cultured cells.