DNA can form structures other than the Watson-Crick double helix. The potential contributions to gene regulation from one such structure have been investigated by assembling a promoter capable of adopting cruciform base-pairing. Transcription from this promoter by RNA polymerase in vitro was repressed as the cruciform was extruded by increasing negative DNA supercoiling. Transcription in vivo was induced as supercoiling was relaxed by growth in conditions that inhibit DNA gyrase. A DNA conformational change is therefore capable of regulating the initiation of transcription.