We studied the formation of d(A-T) n cruciforms in E.coli cells by probing intracellular plasmid DNA with chloroacetaldehyde followed by fine analysis of modified DNA bases. d(A-T) 16 sequences were inserted into specifically designed plasmids either upstream of a single trc promoter, or between two divergent trc promoters. We found that in both cases, induction of transcription by IPTG leads to the transition of the d(A-T) 16 stretch into a cruciform state. In the case of two divergent promoters, we observed cruciform formation even without IPTG. Enhanced cruciform formation correlates with the elevation in promoter activity as defined by the opening of the promoter at the ≈10 to +2 positions. We conclude that transcriptionally driven negative supercoiling provokes cruciform formation in vivo