The fraction DE-B obtained by fractionating an extract from rat mammary adenocarcinoma cells on a DEAE-Sephadex column was used for transcribing linear and supercoiled rRNA gene (rDNA). This fraction, which is known to contain RNA polymerase I and essential transcription factors, also contains DNA topoisomerase I activity. Inhibition of this topoisomerase activity by the selective inhibitor camptothecin markedly diminished transcription of supercoiled rDNA, and at a concentration of 150 microM, camptothecin almost completely inhibited DNA topoisomerase I activity and supercoiled rDNA transcription. Addition of exogenous calf thymus DNA topoisomerase I to the sample containing the drug restored the ability of the extract to transcribe supercoiled rDNA. Camptothecin, even at a concentration of 500 microM, had no significant effect on the transcription of linear rDNA. These studies show that relaxation of supercoiled rDNA by DNA topoisomerase I is essential for its transcription. The preferential inhibition of rRNA synthesis in vivo following treatment with camptothecin is probably due to selective camptothecin inhibition of DNA topoisomerase I activity.