Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.