A DNA fragment encompassing the Saccharomyces cerevisiae GAL1--GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase. We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA. Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand-specific endonuclease Pl. In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined. Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS). In vitro transcription with purified S. cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences.