Transcription elongation causes a local change in DNA superhelicity. An excess of negative supercoiling may lead to opening of DNA strands that could allow formation of R-loops. In yeast, mutants of the THO complex are impaired in transcription elongation and this defect has been linked to co-transcriptional formation of R-loops, which could constitute roadblocks for RNA polymerases. In this study, we found that stably formed 300-nt long DNA-RNA hybrids in a negatively supercoiled transcription template reduced the efficiency of transcription elongation by half, providing a first experimental evidence that transcription elongation is impaired by R-loops in vitro.