To elucidate the molecular mechanism by which the potent anticancer drug, 5-fluorouracil (5-FUra), inhibits cell proliferation, the effect of its metabolite, 5-fluorouridine triphosphate, on transcription of rat rRNA gene and processing of pre-rRNA was investigated in S-100 extract from the mouse lymphosarcoma cells. The in vitro processing of pre-rRNA substrate synthesized from the T3 promoter occurred at the correct primary processing site. Replacement of UMP with 5-fluorouridine monophosphate in the rRNA substrate did not affect the pre-rRNA processing. Similar result was obtained when coupled transcription-processing was studied. When the coupled reaction was examined using extracts from the cells treated with 5-FUra, rRNA processing was abolished whereas transcription of rRNA gene was unaffected. Treatment of cells with thymidine along with 5-FUra did not reverse the inhibitory effect of the drug on rRNA processing. In contrast to the effect on rRNA processing, treatment of cells with 5-FUra did not impede the 3' end processing of pre-mRNA. These data show that inhibition of pre-rRNA processing is a major mechanism of action of 5-FUra and suggest that the activity and/or synthesis of a trans-acting factor(s) involved in this reaction is altered by the anticancer drug.