Novobiocin has been shown to inhibit class III gene transcription from both chromatin and non-chromatin templates. Since novobiocin is a well characterized inhibitor of type II DNA topoisomerases, it has been postulated that a gyrase activity is necessary for transcription. Using DNase I footprinting, we show here that novobiocin inhibits the specific binding of polymerase III transcription factors TFIIIA and TFIIIC to the promoters of the 5S RNA and VA RNA genes, respectively. Concentrations of novobiocin employed were comparable to those necessary to inhibit HeLa topoisomerase II. In vitro transcription assays, performed under equivalent conditions, demonstrated that similar novobiocin concentrations were necessary for transcription inhibition. These results strongly suggest that novobiocin interferes with transcription by inhibiting specific protein-DNA interactions.