Polypurine-polypyrimidine (R.Y) sequences have the unusual ability to form DNA triple helices. Such tracts are overrepresented upstream of eukaryotic genes, although a function there has not been clear. We report that transcription in vitro into one such upstream R.Y tract in the direction that makes a predominantly purine RNA is effectively blocked by formation of an intramolecular triple helix. The triplex is triggered by transcription and stabilized by the binding of nascent purine RNA to the template. Transcription in the opposite direction is not restricted. Polypurine-polypyrimidine DNA may provide a dynamic and selective block to transcription without the aid of accessory proteins.