DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G-quartets; planar arrays of four hydrogen-bonded guanines. It was recently reported that G4 DNA can be generated in E. coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ+ strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we have studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We have used a reconstituted transcription system in vitro with purified polymerase and initiation factors, and with substrates containing G-rich sequences in either the transcribed or the non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase but that when located in the non-transcribed strand they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared to single events.