We show that transcription of a DNA molecule inside a bacterium is accompanied by local and temporal supercoiling of the DNA template: as transcription proceeds, DNA in front of the transcription ensemble becomes positively supercoiled, and DNA behind the ensemble becomes negatively supercoiled. Because bacterial gyrase and topoisomerase I act differently on positively and negatively supercoiled DNA, the formation of twin supercoiled domains during transcription is manifested by a large increase or decrease in the linking number of an intracellular plasmid when bacterial DNA gyrase or topoisomerase I, respectively, is inhibited. Such changes in linking number are strongly dependent on transcription of the plasmid in cis and on the relative orientations of transcription units on the plasmid. These results indicate that the state of supercoiling of bacterial DNA is strongly modulated by transcription, and that DNA topoisomerases are normally involved in the elongation step of transcription.