The bgl operon of Escherichia coli is transcriptionally inactive in wild-type cells. DNA insertion sequences (IS) constitute a major class of spontaneous mutations that activate the cryptic bgl promoter. In an attempt to study the molecular mechanism of activation mediated by insertion sequences, transcription of the bgl promoter was carried out in vitro. Stimulation of transcription is observed when a plasmid containing an insertionally activated bgl promoter is used as a template in the absence of proteins other than RNA polymerase. Deletions that remove sequences upstream of the bgl promoter, and insertion of a 1.2 kb DNA fragment encoding resistance to kanamycin, activate the promoter. Point mutations within a region of dyad symmetry upstream of the promoter, which has the potential to extrude into a cruciform structure under torsional stress, also lead to activation. Introduction of a sequence with dyad symmetry, upstream of an activated bgl promoter carrying a deletion of upstream sequences, results in a fourfold reduction in transcription. These results suggest that the cryptic nature of the bgl promoter is because of the presence of DNA structural elements near the promoter that negatively affect transcription.