The effect of drugs which inhibit DNA gyrase, including nalidixic acid, oxolinic acid and coumerycin, on transcription of Escherichia coli bacteria, phage and plasmid genomes was studied. Quantitative estimates of the synthesis of RNA under drug-treatment conditions showed that synthesis of many RNA species, including trp mRNA, was subject to inhibiton by the drug. Transcription directed by the lambda promoter pR was selectively less sensitive to the drug action than transcription initiated at the lambda promoter pL. Evidence was obtained showing that diminished transcription resulted from less frequent RNA chain initiation rather than a premature arrest of the chain elongation. Inhibiton of transcription by these DNA gyrase inhibitors was observed even in the absence of DNA replication. The inhibition by oxolinic acid or coumerycin was not observed in an E. coli strain bearing a nalAr mutation or a cour mutation, respectively. The reduction of trp mRNA synthesis in oxolinic acid-treated cells cannot be attributed to the increase in the rate of nascent mRNA degradation. These results indicate that DNA gyrase is generally required for intracellular RNA synthesis, and suggest that the supercoiling of DNA by this winding enzyme enhances the initiation of transcription.