Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.